Compositions and methods for producing platelets and/or proplatelets from megakaryocytes

ABSTRACT

The present invention describes novel compositions and methods to enhance the in vitro and in vivo production of platelets and/or proplatelets from megakaryocytes. The present invention describes compositions comprising megakaryocytes, nitric oxide donors (i.e. compounds that donate, transfer or release nitric oxide, elevate endogenous levels of endothelium-derived relaxing factor, stimulate endogenous synthesis of nitric oxide or are substrates for nitric oxide synthase), and, optionally, at least one thrombopoiesis stimulating factor. The thrombopoiesis stimulating factor is preferably thrombopoietin. The nitric oxide donor is preferable S-nitrosoglutathione. The present invention also describes compositions comprising at least one nitric oxide donor and at least one thrombopoiesis stimulating factor. The present invention also provides methods for treating and/or preventing blood platelet disorders, and for producing platelets and/or proplatelets in vitro and in vivo. The compounds and/or compositions of the present invention can be provided in the form of a pharmaceutical kit.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/126,854 filed Mar. 30, 1999.

FIELD OF THE INVENTION

The present invention describes in vitro and in vivo production ofplatelets and/or proplatelets from megakaryocytes. The present inventionis also directed to compositions comprising megakaryocytes, nitric oxidedonors (i.e. compounds that donate, transfer or release nitric oxide,elevate endogenous levels of endothelium-derived relaxing factor,stimulate endogenous synthesis of nitric oxide or are substrates fornitric oxide synthase), and, optionally, at least one thrombopoiesisstimulating factor. The thrombopoiesis stimulating factor is preferablythrombopoietin. The nitric oxide donor is preferableS-nitrosoglutathione. The present invention also describes compositionscomprising at least one nitric oxide donor and at least onethrombopoiesis stimulating factor. The present invention also providesmethods for treating and/or preventing blood platelet disorders, and forproducing platelets and/or proplatelets in vitro and in vivo. Thecompounds and/or compositions of the present invention can be providedin the form of a pharmaceutical kit.

BACKGROUND OF THE INVENTION

Platelets are circulating cell derived fragments that are required forthe maintenance of hemostasis. These small, anucleate fragmentsrepresent the first line of defense against hemorrhage followingvascular injury, and are crucial for blood coagulation. Platelets arethe terminal differentiation product of megakaryocytes, which in turnoriginate from pluripotent stem cells. The process of plateletproduction from megakaryocytes, which is complex and incompletelyunderstood, is called thrombopoiesis. Several cytokines have beenreported to stimulate the growth and maturation of megakaryocytes. Theinteraction between the cytokines and growth factors, their kineticchoreography, and the specific molecular steps that commit themegakaryocytes and their precursors to the process of maturation andplatelet production have only begun to be rigorously investigated.Megakaryocytes mature by a process of endomitosis and cytoplasmicmaturation. Most research to date has focused on the maturation step ofmegakaryocyte growth rather than on the terminal process of plateletproduction.

Morphological studies of marrow megakaryocytes suggest that plateletsform as a result of cytoplasmic fragmentation. With the completion ofendomitosis, megakaryocyte cytoplasm expands and, in the process,develops demarcation membranes and granules. Platelets form as the fullymature megakaryocyte develops cytoplasmic extensions, or pseudopodialprotrusions, that extend in proximity to sinusoidal endothelial cells(Tavassoli and Aoki, Blood Cells, 15:3-14, (1989)). Platelets bud fromthe ends of these protusions and thereafter enter the circulation. Themegakaryocyte's ability to produce platelet buds is ultimatelyexhausted, and it undergoes terminal apoptosis.

The in vitro counterpart to thrombopoiesis is believed to be thedevelopment of the “proplatelet” process that has been observed in theterminal phases of megakaryocyte tissue cultures (Choi et al, Blood,85:402-413 (1995)). Some data suggests that proplatelets can produceplatelet-like particles (Choi et al, Blood, 402-413 (1995); Zeigler etal, Blood, 84:4045-4052 (1994)). Proplatelets insinuating between bonemarrow sinusoidal cells can enter the circulation (Tavassoli et al,Blood Cells, 15:3-14 (1989)). Circulatory shear forces within the marrowor possibly in the pulmonary circulation could result in thefragmentation of these proplatelets, thereby producing platelets incirculation (Burstein et al, Magakaryopoiesis and Platelet Formation,McGraw-Hill, New York, (1995); Trowbridge et al, Thromb Res., 28:461-475(1982)).

A number of diseases or conditions result from inappropriate levels orinadequate functioning of blood platelets. Platelet disorders areclinically treated by administering thrombopoietin or by whole blood orplatelet transfusions. Platelets for such procedures are obtained byplateletphoresis from normal donors; however, blood and plateletsupplies can be limited. In addition, platelets have a relatively shortshelf-life of about 5 days. Transfusions are also costly and cantransmit infections and expose patients to viruses such as the humanimmunodeficiency virus (HI) or various hepatitis viruses. Furthermore,patients are often refractory to subsequent transfusions. Thrombopoietintreatment has a lag period before the level of platelets are affectedand often results in the failure to stimulate platelet production inmany patients.

Thus, there remains a need in the art for new and improved methods forin vitro production of platelets for use by patients and for new andimproved methods of stimulating or enhancing the production of plateletsin vivo, thereby resulting in safer alternatives for treating and/orpreventing blood platelet disorders. The present invention is directedto these, as well as other, important ends.

SUMMARY OF THE INVENTION

Nitric oxide (NO) has been shown to mediate a number of actions,including the bactericidal and actions of macrophages and blood vesselrelaxation of endothelial cells. NO and NO donors have also beenimplicated as mediators for a number of processes includingvasodilation, neurotransmission, immunity, and vascular and nonvascularsmooth muscle relaxation. In the process of arriving at the presentinvention, it was hypothesized that when the megakaryocytes arecommitted to platelet production, an endothelial product is releasedfrom marrow sinusoidal endothelial cells as they make contact withmegakaryocyte protrusions. This endothelium product should promoteapoptosis, since platelet formation morphologically resembles programmedcell death (Radley and Hailer, Br. J. Haematol., 53:227-287 (1983)). Onepossible endothelial product that promotes apoptosis is nitric oxide.

One aspect of the present invention provides compositions comprising atleast one megakaryocyte and at least one compound that donates,transfers or releases nitrogen monoxide as a charged species, i.e.,nitrosonium (NO⁺) or nitroxyl (NO−), or as the neutral species, nitricoxide (NO·), and/or stimulates endogenous production of nitric oxide orendothelium-derived relaxing factor (EDRF) in vivo and/or is a substratefor nitric oxide synthase (i.e., nitric oxide donor). Preferably, thenitric oxide donor is S-nitroso-glutathione. The compositions cancomprise a pharmaceutically acceptable carrier. These compositions,which are useful for the in vitro production of platelets and/orproplatelets, can be produced by treating megakaryocytes in culture withan effective amount of at least one nitric oxide donor. Thesecompositions potentiate apoptosis of the cells and increase the numberof platelets and/or proplatelets produced.

Another aspect of the present invention provides compositions comprisingat least one megakaryocyte, at least one thrombopoiesis stimulatingfactor, and at least one compound that donates, transfers or releasesnitrogen monoxide as a charged species, i.e., nitrosonium (NO⁺) ornitroxyl (NO−), or as the neutral species, nitric oxide (NO·), and/orstimulates endogenous production of nitric oxide or EDRF in vivo and/oris a substrate for nitric oxide synthase. The thrombopoiesis stimulatingfactor is preferably thrombopoietin (TPO), and the nitric oxide donor ispreferably S-nitroso-glutathione. The compositions can comprise apharmaceutically acceptable carrier. These compositions, which areuseful for the in vitro production of platelets and/or proplatelets, canbe produced by treating megakaryocytes in culture with an effectiveamount of at least one thrombopoiesis stimulating factor and at leastone nitric oxide donor. Preferably, the compositions are produced bytreating megakaryocytes in culture with an effective amount of at leastone thrombopoiesis stimulating factor followed by treatment of the cellswith at least one nitric oxide donor. These compositions increase theproduction of platelets and/or proplatelets in culture.

Another aspect of the present invention provides methods for stimulatingthe in vivo production of a patient's own platelets and/or proplateletsby administering to a patient a therapeutically effective amount of atleast one thrombopoiesis stimulating factor and at least one compoundthat donates, transfers or releases nitrogen monoxide as a chargedspecies, i.e., nitrosonium (NO⁺) or nitroxyl (NO−), or as the neutralspecies, nitric oxide (NO·), and/or stimulates endogenous production ofnitric oxide or EDRF in vivo and/or is a substrate for nitric oxidesynthase, thereby augmenting the production of the patient's ownplatelets and/or proplatelets. The thrombopoiesis stimulating factor andnitric oxide donor can be administered separately or as components ofthe same composition in one or more pharmaceutically acceptablecarriers.

Another aspect of the present invention provides methods for treatingand/or preventing blood platelet disorders in a patient by administeringa therapeutically effective amount of at least one compound thatdonates, transfers or releases nitrogen monoxide as a charged species,i.e., nitrosonium (NO⁺) or nitroxyl (NO−), or as the neutral species,nitric oxide (NO·), and/or stimulates endogenous production of nitricoxide or EDRF in vivo and/or is a substrate for nitric oxide synthase,and, optionally, at least one thrombopoiesis stimulating factor. Thethrombopoiesis stimulating factor and nitric oxide donor can beadministered separately or as components of the same composition in oneor more pharmaceutically acceptable carriers. The nitric oxide donorand, optionally, at least one thrombopoiesis stimulating factor can alsobe administered in combination with other medications for the treatmentof blood platelet disorders. These methods of treating and/or preventingplatelet disorders are preferable to current transfusion therapies whichare vehicles for infections, such as AIDS and hepatitis.

Another aspect of the present invention provides methods for treatingand/or preventing blood platelet disorders in a patient by administeringplatelets and/or proplatelets produced by culturing megakaryocytes invitro. The megakaryocytes in culture are treated with an effectiveamount of at least one compound that donates, transfers or releasesnitrogen monoxide as a charged species, i.e., nitrosonium (NO⁺) ornitroxyl (NO−), or as the neutral species, nitric oxide (NO·), and/orstimulates endogenous production of nitric oxide or EDRF in vivo and/oris a substrate for nitric oxide synthase, and, optionally, at least onethrombopoiesis stimulating factor. The thrombopoiesis stimulating factorand nitric oxide donor can be administered separately or as componentsof the same composition in one or more pharmaceutically acceptablecarriers. The megakaryocytes in culture can be derived from a variety ofsources, such as cell lines, stem cells, tissues, bone marrow or apatient's own blood megakaryocytic precursors. These methods of treatingplatelet disorders are preferable to current transfusion therapies whichare vehicles for infections, such as AIDS and hepatitis.

Yet another aspect of the present invention provides methods forreducing high platelet counts in a patient by administering an effectiveamount of at least one compound that inhibits the patient's productionof nitric oxide.

These and other aspects of the present invention are described in detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D are flow cytometric analyses of human platelet glycoproteinIIIa (GPIIIa) positive platelet sized particles from Example 1. FIG. 1Ashows an untreated cultured sample of Meg-01 cells positive for GPIHIa.FIG. 1B shows cultured Meg-01 cells treated with 100 μMS-nitroso-glutathione (SNO-glu) and the formation of GPIIIa positiveplatelet-sized particle. FIG. 1C shows cultured Meg-01 cells pretreatedwith 100 ng/ml thrombopoietin, washed with phosphate-buffered saline,and treated with 100 μM SNO-glu. FIG. 1D shows aggregation of plateletrich fraction with 10 μM thrombin-receptor activating protein (Trap),2.5 mM CaCl₂, and 600 μg/ml fibrinogen.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings.

“Patient” refers to animals, preferably mammals, more preferably humans.

“Proplatelets” refer to any structural form of a megakaryocyte or itsfragments, such as cytoplasmically-linked platelet-like particles, thatcould result in platelet formation. The structural forms include, butare not limited to, cells with long cytoplasmic extensions, projectionsor pseudopodia that contain swellings encompassing platelet bodies invarious stages of formation, such as, nodules, blebs, and the like.

“Blood platelet disorder” refers to a condition or disorder caused byblood platelet dysfunction or an insufficient or over supply of bloodplatelets. Exemplary blood platelet disorders include thrombocytopenia,thrombocythemia and thrombocytopathy.

“Thrombocytopenia” refers to blood platelet disorders characterized bylow platelet counts. Exemplary thrombocytopenic platelet disorders areautoimmune, neonatal thrombocytopenia; thrombotic thrombocytopenicpurpura; idiopathic (immune) thrombocytopenic purpura; dilutionalthrombocytopenia; low platelet count conditions resulting from orassociated with aplastic anemia; malignant infiltration; chemotherapy orother bone marrow failure states; bone marrow transplantation;antibody-mediated platelet destruction; blood transfusion;cardiopulmonary by-pass; AIDS; disseminated intravascular coagulation;hemolytic uremic syndrome; leukemia; hypersplenism; myelodysplasticdisorders and arteriovenous realformations; pulmonary hypertension;kidney graft rejection; and administration of heparin or certain otherdrugs.

“Thrombocythemia” refers to blood platelet disorders characterized by arelatively low platelet count. Exemplary thrombocythemic plateletdisorders are idiopathic thrombocythemia and high platelet countsresulting from or associated with reactive thrombocytosis secondary toinflammation; iron deficiency or malignancy, polycythemia vera; chronicmyelogenous leukemia; myeloid metaplasia or any other myeloproliferativecondition.

“Thrombocytopathy” refers to blood platelet disorders characterized byan abnormally high or low platelet function, although the plateletcounts can be in the normal range. Exemplary thrombocytopathic disordersin which the platelet function is low include Mediterraneanthrombocytopathy; von Willebrand's disease; and idiopathic (immune)thrombocytopenic purpura. Low platelet function thrombocytopathicconditions can also be associated with or result from HIV infections;drug induced or hereditary storage pool disorders; uremia; andmyelodysplastic disorders or thrombolytic therapy. Exemplarythrombocytopathic disorders in which the platelet function is highinclude thrombocythemia. Thrombocytopathic conditions can also beassociated with or result from myeloproliferative disorders;atherosclerosis; myocardial infraction; unstable angina; stroke andother vascular thrombosis disorders such as peripheral vasculardisorders and the like.

“Thrombopoiesis stimulating factors” refer to components that arecapable of stimulating the growth and maturation of megakaryocytes orhematopoietic stem cells, including cytokines and growth factors.Exemplary thrombopoiesis stimulating factors are interleukins (IL) 1 to15 (preferably IL-3, IL-6 and IL-11), erythropoietin (EPO),thrombopoietin (TPO), stem cell factors (SCF, also known as mast cellgrowth factor and c-kit ligand), flt-3 ligand (FL), granulocyte colonystimulating factor (GCSF), granulocyte macrophage colony stimulatingfactor (GM-CSF), tumor growth factor beta (TGF beta), tumor necrosisfactor alpha (TNF alpha), interferon (IFN alpha, beta or gamma),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),insulin-like growth factors (IGF-1 and IGF-2), leukemia inhibitor factor(LIF), megakaryoctye colony stimulating factor (meg-CSF) and the like.Thrombopoiesis stimulating factors are commercially available, forexample, from R&D Systems (Minneapolis, Minn.) or they can be chemicallysynthesized or isolated by extraction and purification from naturalsources, from recombinant cell cultures or recombinant DNA methods.Biologically active equivalents of thrombopoiesis stimulating factorsare also effective in stimulating the growth and maturation ofmegakaryocytes. Biologically active equivalents of thrombopoiesisstimulating factors include, for example, those differing in one or moreamino acids in the overall sequence or in the glycosylation pattern;substituted, deleted and/or inserted amino acid variants; and/or posttranslation modified factors.

“Carriers” or “vehicles” refers to carrier materials suitable forcompound administration and include any such material known in the artsuch as, for example, any liquid, gel, solvent, liquid diluent,solubilizer, or the like, which is non-toxic and which does not interactwith any components of the composition in a deleterious manner.

“Nitric oxide adduct” or “NO adduct” refers to compounds and functionalgroups which, under physiological conditions, can donate, release and/ordirectly or indirectly transfer any of the three redox forms of nitrogenmonoxide (NO⁺, NO⁻, NO·), such that the biological activity of thenitrogen monoxide species is expressed at the intended site of action.

“Nitric oxide releasing” or “nitric oxide donating” refers to methods ofdonating, releasing and/or directly or indirectly transferring any ofthe three redox forms of nitrogen monoxide (NO⁺, NO−, NO·), such thatthe biological activity of the nitrogen monoxide species is expressed atthe intended site of action.

“Nitric oxide donor” or “NO donor” refers to compounds that donate,release and/or directly or indirectly transfer a nitrogen monoxidespecies, and/or stimulate the endogenous production of nitric oxide orendothelium-derived relaxing factor (EDRF) in vivo and/or elevateendogenous levels of nitric oxide or EDRF in vivo. “NO donor” alsoincludes compounds that are substrates for nitric oxide synthase.

“Alkyl” refers to a lower alkyl group, a haloalkyl group, an alkenylgroup, an alkynyl group, a bridged cydoalkyl group, a cycloalkyl groupor a heterocyclic ring, as defined herein.

“Lower alkyl” refers to branched or straight chain acyclic alkyl groupcomprising one to about ten carbon atoms (preferably one to about eightcarbon atoms, more preferably one to about six carbon atoms). Exemplarylower alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl, hexyl, octyl,and the like.

“Haloalkyl” refers to a lower alkyl group, an alkenyl group, an alkynylgroup, a bridged cycloalkyl group, a cycloalkyl group or a heterocyclicring, as defined herein, to which is appended one or more halogens, asdefined herein.

Exemplary haloalkyl groups include trifluoromethyl, chloromethyl,2-bromobutyl, 1-bromo-2-chloro-pentyl, and the like.

“Alkenyl” refers to a branched or straight chain C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₆ hydrocarbon)which can comprise one or more carbon-carbon double bonds. Exemplaryalkenyl groups include propylenyl, buten-1-yl, isobutenyl, penten-1-yl,2,2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, hepten-1-yl,octen-1-yl, and the like.

“Alkynyl” refers to an unsaturated acyclic C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₆ hydrocarbon)which can comprise one or more carbon-carbon triple bonds. Exemplaryalkynyl groups include ethynyl, propynyl, butyn-1-yl, butyn-2-yl,pentyl-1-yl, pentyl-2-yl, 3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl,hexyl-3-yl, 3,3-dimethyl-butyn-1-yl, and the like.

“Bridged cydoalkyl” refers to two or more cycloalkyl groups,heterocyclic groups, or a combination thereof fused via adjacent ornon-adjacent atoms. Bridged cydoalkyl groups can be unsubstituted orsubstituted with one, two or three substituents independently selectedfrom alkyl, alkoxy, amino, alkylamino, dialkylamnino, hydroxy, halo,carboxyl, alkylcarboxylic acid, aryl, amidyl, ester, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo and nitro. Exemplary bridgedcydoalkyl groups include adamantyl, decahydronapthyl, quinuclidyl,2,6-dioxabicydo[3.3.0]octane, 7-oxabycyclo[2.2.1]heptyl,8-azabicyclo[3,2,1]oct-2-enyl and the like.

“Cydoalkyl” refers to a saturated or unsaturated cyclic hydrocarboncomprising from about 3 to about 8 carbon atoms. Cydoalkyl groups can beunsubstituted or substituted with one, two or three substituentsindependently selected from alkyl, alkoxy, amino, alkylamino,dialkylamino, arylamino, diarylamino, alkylarylamino, aryl, amidyl,ester, hydroxy, halo, carboxyl, alkylcarboxylic acid, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo and nitro. Exemplarycycloalkyl groups include cydopropyl, cyclobutyl, cyclopentyl,cydohexyl, cyclohexenyl, cydohepta,1,3ienyl, and the like.

“Heterocyclic ring or group” refers to a saturated, unsaturated, cyclicor aromatic or polycyclic hydrocarbon group having about 3 to about 12carbon atoms (preferably about 4 to about 6 carbon atoms) where 1 toabout 4 carbon atoms are replaced by one or more nitrogen, oxygen and/orsulfur atoms. Sulfur maybe in the thio, sulfinyl or sulfonyl oxidationstate. The heterocyclic ring or group can be fused to an aromatichydrocarbon group. Heterocyclic groups can be unsubstituted orsubstituted with one, two or three substituents independently selectedfrom alkyl, alkoxy, amino, alkylamino, dialkylamino, arylamino,diarylamino, alkylarylamino, hydroxy, oxo, thial, halo, carboxyl,carboxylic ester, alkylcarboxylic acid, alkylcarboxylic ester, aryl,arylcarboxylic acid, arylcarboxylic ester, amidyl, ester, carboxamido,alkylcarboxamido, arylcarboxamido, sulfonic acid, sulfonic ester,sulfonamido and nitro. Exemplary heterocyclic groups include pyrrolyl,3-pyrrolinyl,4,5,6-trihydro-2H-pyranyl, pyridinyl, 1,4-dihydropyridinyl,pyrazolyl, triazolyl, pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl,imidazolyl, indolyl, thiophenyl, furanyl, tetrhydrofuranyl, tetrazolyl,2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, oxazolindinyl 1,3-dioxolanyl,2,6-dioxabicydo[3,3,0]octanyl, 2-imidazonlinyl, imidazolindinyl,2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl,1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl,4H-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl,1,3,5-trithianyl, benzo(b)thiophenyl, benzimidazolyl, quinolinyl, andthe like.

“Heterocyclic compounds” refer to mono- and polycydic compoundscomprising at least one aryl or heterocyclic ring.

“Aryl” refers to a monocyclic, bicyclic, carbocyclic or heterocyclicring system comprising one or two aromatic rings. Exemplary aryl groupsinclude phenyl, pyridyl, napthyl, quinoyl, tetrahydronaphthyl, furanyl,indanyl, indenyl, indoyl, and the like. Aryl groups (including bicylicaryl groups) can be unsubstituted or substituted with one, two or threesubstituents independently selected from alkyl, alkoxy, amino,alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino,hydroxy, carboxyl, carboxylic ester, alkylcarboxylic acid,alkylcarboxylic ester, aryl, arylcarboxylic acid, arylcarboxylic ester,alkylcarbonyl, arylcarbonyl, amidyl, ester, carboxamido,alkylcarboxamido, carbomyl, sulfonic acid, sulfonic ester, sulfonamidoand nitro. Exemplary substituted aryl groups include tetrafluoro-phenyl,pentafluorophenyl, sulfonamide, alkylsulfonyl, arylsulfonyl, and thelike.

“Alkylaryl” refers to an alkyl group, as defined herein, to which isappended an aryl group, as defined herein. Exemplary alkylaryl groupsinclude benzyl, phenylethyl, hydroxybenzyl, fluorobenzyl,fluorophenylethyl, and the like.

“Arylalkyl” refers to an aryl radical, as defined herein, attached to analkyl radical, as defined herein.

“Cycloalkylalkyl” refers to a cycloalkyl radical, as defined herein,attached to an alkyl radical, as defined herein.

“Heterocyclicalkyl” refers to a heterocyclic ring radical, as definedherein, attached to an alkyl radical, as defined herein.

“Cycloalkenyl” refers to an unsaturated cyclic hydrocarbon having about3 to about 10 carbon atoms (preferably about 3 to about 8 carbon atoms,more preferably about 3 to about 6 carbon atoms) comprising one or morecarbon-carbon double bonds.

“Arylheterocyclic ring” refers to a bi- or tricyclic ring comprised ofan aryl ring, as defined herein, appended via two adjacent carbon atomsof the aryl ring to a heterocyclic ring, as defined herein. Exemplaryarylheterocyclic rings include dihydroindole,1,2,3,4-tetra-hydroquinoline, and the like.

“Alkoxy” refers to R₅₀O-, wherein R₅₀ is an alkyl group, as definedherein. Exemplary alkoxy groups include methoxy, ethoxy, t-butoxy,cyclopentyloxy, and the like.

“Arylalkoxy or alkoxyaryl” refers to an alkoxy group, as defined herein,to which is appended an aryl group, as defined herein. Exemplaryarylalkoxy groups indude benzyloxy, phenylethoxy, chlorophenylethoxy,and the like.

“Alkoxyalkyl” refers to an alkoxy group, as defined herein, appended toan alkyl group, as defined herein. Exemplary alkoxyalkyl groups includemethoxymethyl, methoxyethyl, isopropoxymethyl, and the like.

“Alkoxyhaloalkyl” refers to an alkoxy group, as defined herein, appendedto a haloalkyl group, as defined herein. Exemplary alkoxyhaloalkylgroups include 4-methoxy-2-chlorobutyl and the like.

“Cycloalkoxy” refers to R₅₄O-, wherein R₅₄ is a cycloalkyl group or abridged cydoalkyl group, as defined herein. Exemplary cycdoalkoxy groupsinclude cyclopropyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

“Haloalkoxy” refers to a haloalkyl group, as defined herein, to which isappended an alkoxy group, as defined herein. Exemplary haloalkyl groupsinclude 1,1,1-trichloroethoxy, 2-bromobutoxy, and the like.

“Hydroxy” refers to —OH.

“Oxo” refers to =O.

“Oxy” refers to —O ⁻R₇₇ ⁺ wherein R₇₇ is an organic or inorganic cation.

“Organic cation” refers to a positively charged organic ion. Exemplaryorganic cations include alkyl substituted ammonium cations, and thelike.

“Inorganic cation” refers to a positively charged metal ion. Exemplaryinorganic cations include metal cations such as for example, sodium,potassium, calcium, and the like.

“Hydroxyalkyl” refers to a hydroxy group, as defined herein, appended toan alkyl group, as defined herein.

“Amino” refers to —NH₂.

“Nitrate” refers to —O—NO₂.

“Nitrite” refers to —O—NO.

“Thionitrate” refers to —S—NO₂.

“Thionitrite” and “nitrosothiol” refer to —S—NO.

“Nitro” refers to the group —NO₂ and “nitrosated” refers to compoundsthat have been substituted therewith.

“Nitroso” refers to the group —NO and “nitrosylated” refers to compoundsthat have been substituted therewith.

“Nitrile” and “cyano” refer to —CN.

“Halogen” or “halo” refers to iodine (I), bromine (Br), chlorine (Cl),and/or fluorine (F).

“Alkylamino” refers to R_(═)NH—, wherein R_(═)is an alkyl group, asdefined herein. Exemplary alkylamino groups include methylamino,ethylamino, butylamino, cydohexylamino, and the like.

“Arylamino” refers to R₅₅NH—, wherein R₅₅is an aryl group, as definedherein.

“Dialkylamino” refers to R₅₀R₅₂N—, wherein R₅₀ and R₅₂ are eachindependently an alkyl group, as defined herein. Exemplary dialkylaminogroups include dimethylamino, diethylamino, methyl propargylamino, andthe like.

“Diarylamino” refers to R₅₅R₆₀N—, wherein R₅₅ and R₆₀ are eachindependently an aryl group, as defined herein.

“Alkylarylamino” refers to R₅₀R₅₅N—, wherein R₅₀ is an alkyl group, asdefined herein, and R₅₅ is an aryl group, as defined herein.

“Aminoalkyl” refers to an amino group, an alkylamino group, adialkylamino group, an arylamino group, a diarylamino group, analkylarylamino group or a heterocyclic ring, as defined herein, to whichis appended an alkyl group, as defined herein.

“Aminoaryl” refers to an amino group, an alkylamino group, adialkylamino group, an arylamino group, a diarylamino group, analkylarylamino group or a heterocyclic ring, as defined herein, to whichis appended an aryl group, as defined herein.

“Thio” refers to —S—.

“Sulfinyl” refers to —S(O)—.

“Methanthial” refers to —C(S)—.

“Thial” refers to =S.

“Sulfonyl” refers to —S(O)₂.

“Sulfonic acid” refers to —S(O)₂OR₇₆, wherein R₇₆ is a hydrogen, anorganic cation or an inorganic cation.

“Alkylsulfonic acid” refers to a sulfonic acid group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonic acid” refers to an sulfonic acid group, as defined herein,appended to an aryl group, as defined herein

“Sulfonic ester” refers to —S(O)₂OR₅₈, wherein R₅₈ is an alkyl group, anaryl group, an alkylaryl group or an aryl heterocyclic ring, as definedherein.

“Sulfonamido” refers to —S(O)₂—N(R₅₁)(R₅₇), wherein R₅₁, and R₅₇ areeach independently a hydrogen atom, an alkyl group, an aryl group, analkylaryl group, or an arylheterocyclic ring, as defined herein, andR₅₁, and R₅₇ when taken together are a heterocyclic ring, a cycloalkylgroup or a bridged cycloalkyl group, as defined herein.

“Alkylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an aryl group, as defined herein.

“Alkylthio” refers to R₅₀S—, wherein R₅₀ is an alkyl group, as definedherein.

“Arylthio” refers to R₅₅S—, wherein R₅₅ is an aryl group, as definedherein.

“Cycloalkylthio” refers to R₅₄S—, wherein R₅₄ is a cydoalkyl group or abridged cycloalkyl group, as defined herein. Exemplary cycloalkylthiogroups include cyclopropylthio, cyclopentylthio, cyclohexylthio, and thelike.

“Alkylsulfinyl” refers to R₅₀—S(O)—, wherein R₅₀ is an alkyl group, asdefined herein.

“Alkylsulfonyl” refers to R₅₀—S(O)₂—, wherein R₅₀ is an alkyl group, asdefined herein.

“Arylsulfinyl” refers to R₅₅—S(O)—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylsulfonyl” refers to R₅₅—S(O)₂—, wherein R₅₅is an aryl group, asdefined herein.

“Amidyl” refers to R₅C(O)N(R₅₇)— wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group, analkylaryl group, or an aryiheterocydic ring, as defined herein.

“Ester” refers to R₅C(O)O— wherein R₅₁ is a hydrogen atom, an alkylgroup, an aryl group, an alkylaryl group, or an arylheterocycdic ring,as defined herein.

“Carbamoyl” refers to —O—C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group, analkylaryl group or an arylheterocyclic ring, as defined herein, or R₅₁and R₅₇ taken together are a heterocyclic ring, a cycloalkyl group or abridged cydoalkyl group, as defined herein.

“Carbamate” refers to R₅₁O—C(O)N(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group, analkylaryl group or an arylheterocyclic ring, as defined herein, or R₅₁and R₅₇ taken together are a heterocydic ring, a cycloalkyl group or abridged cydoalkyl group, as defined herein.

“Carboxyl” refers to —C(O)OR₇₆, wherein R₇₆ is a hydrogen, an organiccation or an inorganic cation, as defined herein.

“Carbonyl” refers to —C(O)—.

“Alkylcarbonyl” or “alkanoyl” refers to R₅₀ —C(O)—, wherein R₅₀ is analkyl group, as defined herein.

“Arylcarbonyl” or “aroyl” refers to R₅₅—C(O)—, wherein R₅₅ is an arylgroup, as defined herein.

“Carboxylic ester” refers to —C(O)OR_(58,) wherein R₅₈ is an alkylgroup, an aryl group, an alkylaryl group or an aryl heterocyclic ring,as defined herein.

“Alkylcarboxylic acid” and “alkylcarboxyl” refer to an alkyl group, asdefined herein, appended to a carboxyl group, as defined herein.

“Alkylcarboxylic ester” refers to an alkyl group, as defined herein,appended to a carboxylic ester group, as defined herein.

“Arylcarboxylic acid” refers to an aryl group, as defined herein,appended to a carboxyl group, as defined herein.

“Arylcarboxylic ester” and “arylcarboxyl” refer to an aryl group, asdefined herein, appended to a carboxylic ester group, as defined herein.

“Carboxamido” refers to —C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group, analkylaryl group or an arylheterocyclic ring, as defined herein, and R₅₁and R₅₇ when taken together with the nitrogen to which they are attachedform a heterocyclic ring, a cycloalkyl group or a bridged cycloalkylgroup, as defined herein.

“Alkylcarboxamido” refers to an alkyl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Arylcarboxamnido” refers to an aryl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Urea” refers to —N(R₅₉)—C(O)N(R₅₁)(R₅₇) wherein R₅₁, R_(57,) and R₅₉are each independently a hydrogen atom, an alkyl group, an aryl group,an alkylaryl group, or an arylheterocyclic ring, as defined herein, orR₅₁ and R₅₇ taken together with the nitrogen to which they are attachedform a heterocyclic ring, as defined herein.

“Phosphoryl” refers to —P(R₇₀)(R₇₁)(R₇₂), wherein R₇₀ is a lone pair ofelectrons, sulfur or oxygen, and R₇₁ and R₇₂ are each independently acovalent bond, a hydrogen, a lower alkyl, an alkoxy, an alkylamino, ahydroxy or an aryl, as defined herein.

The present invention provides methods for treating and/or preventingblood platelet disorders by administering the compositions describedherein.

In arriving at the present invention, it was unexpectedly discoveredthat the administration of one or more nitric oxide donors, and theoptional administration of at least one thrombopoiesis stimulatingfactor, to megakaryocytes, increases the in vitro and in vivo plateletand/or proplatelet production. The stimulation or enhancement ofplatelet production solely using thrombopoiesis stimulating factors hasbeen previously described. For example, U.S. Pat. No. 5,571,686describes the use of megapoietin protein for stimulating an increase inthe megakaryocyte size and number; U.S. Pat. No. 5,593,666 describes theuse of thrombopoietin for increasing platelet cell counts inthrombocytopenia; U.S. Pat. Nos. 5,178,856, 5,087,448 and 5,032,396describe the use of interleukins to enhance the growth of megakaryocytesor stimulate platelet production; and U.S. Pat. Nos. 5,498,698,5,498,599 and 5,326,558 describe the use of novel thrombopoiesisstimulating factors (the disclosure of each of these patents areincorporated by reference herein in their entirety). There is nosuggestion in the prior art to treat megakaryocytes with a nitric oxidedonor alone, or in combination with thrombopoiesis stimulating factors,to increase platelet and/or proplatelet production.

In the present invention, the megakaryocytes for use in vitro and invivo indude those obtained from any commercially availablemegakaryocyte-producing cell line, such as, for example, the humanmegakaryoblastic leukemia cell lines Meg-01 and Meg-01s; cells culturedfrom any mammalian source such as CD34⁺ cells from human plasma,CD34⁺CD38— hematopoietic cells, human bone marrow CD34⁺ cells, humanfetal liver CD34⁺ cells, GCSF mobilized peripheral blood CD34⁺ cells,human embryonic stem cells, and the like; and dones produced from any ofthe megakaryocytic cell lines using standard doning cell culturingtechniques, induding, but not limited to, serial dilution of suspensioncells and low density plating of the attached and adhered cells. Themegakaryocytes in culture can also include those isolated from apatient's own blood or stem cells or those isolated from tissues such asbone marrow, peripheral blood, liver, fetal liver, and the like.

A principal aspect of the present invention provides novel compositionscomprising megakaryocytes in combination with nitric oxide and/or nitricoxide donors. The term “nitric oxide” encompasses uncharged nitric oxide(NO·) and charged nitrogen monoxide species, preferably charged nitrogenmonoxide species, such as nitrosonium ion (NO⁺) and nitroxyl ion (NO−).The reactive form of nitric oxide can be provided by gaseous nitricoxide. The nitrogen monoxide releasing, delivering or transferringcompounds include any and all such compounds which provide nitrogenmonoxide to its intended site of action in a form active for itsintended purpose. The term “NO adducts” encompasses any nitrogenmonoxide releasing, delivering or transferring compounds, including, forexample, S-nitrosothiols, nitrites, nitrates, S-nitrothiols,sydnonimines, 2-hydroxy-2-nitroso-hydrazines (NONOates),(E)-alkyl-2-[(E)-hydroxyimino]-5-nitro-3-hexene amines or amides,nitrosoamines, furoxans as well as substrates for the endogenous enzymeswhich synthesize nitric oxide. The “NO adducts” can bemono-nitrosylated, poly-nitrosylated, mono-nitrosated and/orpoly-nitrosated or a combination thereof at a variety of naturallysusceptible or artificially provided binding sites for biologicallyactive forms of nitrogen monoxide.

One group of NO adducts is the S-nitrosothiols, which are compounds thatinclude at least one —S—NO group. These compounds includeS-nitroso-polypeptides (the term “polypeptide” includes proteins andpolyamino acids that do not possess an ascertained biological function,and derivatives thereof); S-nitrosylated amino acids (including naturaland synthetic amino acids and their stereoisomers and racemic mixturesand derivatives thereof); S-nitrosylated sugars; S-nitrosylated,modified and unmodified, oligonucleotides (preferably of at least 5, andmore preferably 5-200 nucleotides); straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedS-nitrosylated hydrocarbons; and S-nitroso heterocyclic compounds.S-nitrosothiols and methods for preparing them are described in U.S.Pat. Nos. 5,380,758 and 5,703,073; WO 97/27749; WO 98/19672; and Oae etal, Org. Prep. Proc. Int., 15(3):165-198 (1983), the disclosures of eachof which are incorporated by reference herein in their entirety.

Another embodiment of the present invention is S-nitroso amino acidswhere the nitroso group is linked to a sulfur group of asulfur-containing amino acid or derivative thereof. Such compoundsinclude, for example, S-nitroso-N-acetylcysteine, S-nitroso-captopril,S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine,S-nitroso-cysteine and S-nitroso-glutathiorie.

Suitable S-nitrosylated proteins include thiol-containing proteins(where the NO group is attached to one or more sulfur groups on an aminoacid or amino acid derivative thereof) from various functional classesincluding enzymes, such as tissue-type plasminogen activator (TPA) andcathepsin B; transport proteins, such as lipoproteins; heme proteins,such as hemoglobin and serum albumin; and biologically protectiveproteins, such as immunoglobulins, antibodies and cytokines. Suchnitrosylated proteins are described in WO 93/09806, the disclosure ofwhich is incorporated by reference herein in its entirety. Examplesinclude polynitrosylated albumin where one or more thiol or othernucleophilic centers in the protein are modified.

Other examples of suitable S-nitrosothiols include:

(i) HS(C(R_(e))(R_(f)))_(m)SNO;

(ii) ONS(C(R_(e))(R_(f)))_(m)R_(e); and

(iii) H₂N—CH(CO₂H)—(CH₂)_(m)—C(O)NH—CH(CH₂SNO)—C(O)NH—CH₂—CO₂H;

wherein m is an integer from 2 to 20; R_(e) and R_(f) are eachindependently a hydrogen, an alkyl, a cycloalkoxy, a halogen, a hydroxy,an hydroxyalkyl, an alkoxyalkyl, an arylheterocyclic ring, an alkylaryl,a cycloalkylalkyl, a heterocyclicalkyl, an alkoxy, a haloalkoxy, anamino, an alkylamino, a dialkylamino, an arylamino, a diarylamino, analkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a sulfonic acid, asulfonic ester, an alkylsulfonic acid, an arylsulfonic acid, anarylalkoxy, an alkylthio, an arylthio, a cydoalkylthio, a cydoalkenyl, acyano, an aminoalkyl, an aminoaryl, an aryl, an arylalkyl, an alkylaryl,a carboxamido, a alkylcarboxamido, an arylcarboxamnido, an amidyl, acarboxyl, a carbamoyl, a carbamate, an alkylcarboxylic acid, anarylcarboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, acarboxylic ester, an alkylcarboxylic ester, an arylcarboxylic ester, ahaloalkoxy, a sulfonamido, an alkylsulfonamido, an arylsulfonamido, asulfonic ester, a urea, a phosphoryl, a nitro, —T—O, or(C(R_(e))(R_(f)))_(k)—T—Q, or R_(e) and R_(f) taken together with thecarbons to which they are attached form a carbonyl, a methanthial, aheterocyclic ring, a cycloalkyl group or a bridged cydoalkyl group; Q is—NO or —NO₂ and T is independently a covalent bond, a carbonyl, anoxygen, —S(O)_(o)— or —N(R_(a))R_(i)—, wherein o is an integer from 0 to2, R_(a) is a lone pair of electrons, a hydrogen or an alkyl group;R_(i) is a hydrogen, an alkyl, an aryl, an alkylcarboxylic acid, an arylcarboxylic acid, an alkylcarboxylic ester, an arylcarboxylic ester, analkylcarboxamido, an arylcarboxamido, an alkylaryl, an alkylsulfinyl, analkylsulfonyl, an arylsulfinyl, an arylsulfonyl, a sulfonamido, acarboxamido, a carboxylic ester, an amino alkyl, an amino aryl,—CH₂—C(T—Q)(R_(e))(R_(f)), or —(N₂O₂ ⁻)·M⁺, wherein M⁺ is an organic orinorganic cation; with the proviso that when R_(i) is—CH₂—C(T—Q)(R_(e))(R_(f)) or —(N₂O₂ ⁻)·M⁺; then “—T—Q” can be ahydrogen, an alkyl group, an alkoxyalkyl group, an aminoalkyl group, ahydroxy group or an aryl group.

In cases where R_(e) and R_(f) are a heterocydic ring or R_(e) and R_(f)when taken together with the carbon atoms to which they are attached area heterocyclic ring, then R_(i) can be a substituent on anydisubstituted nitrogen contained within the radical wherein R_(i) is asdefined herein.

Nitrosothiols can be prepared by various methods of synthesis. Ingeneral, the thiol precursor is prepared first, then converted to theS-nitrosothiol derivative by nitrosation of the thiol group with NaNO₂under acidic conditions (pH is about 2.5) which yields the S-nitrosoderivative. Acids which can be used for this purpose include aqueoussulfuric, acetic and hydrochloric acids. The thiol precursor can also benitrosylated by reaction with an organic nitrite such as tert-butylnitrite, or a nitrosonium salt such as nitrosonium tetraflurorborate inan inert solvent.

Another group of NO adducts for use in the present invention, where theNO adduct is a compound that donates, transfers or releases nitricoxide, include compounds comprising at least one ON—O—, ON—N— or ON—C—group. The compounds that include at least one ON—O—, ON—N— or ON—C—group are preferably ON—O—, ON—N— or ON—C-polypeptides (the term“polypeptide” includes proteins and polyamino acids that do not possessan ascertained biological function, and derivatives thereof); ON—O,ON—N— or ON—C-amino acids (including natural and synthetic amino acidsand their stereoisomers and racemic mixtures); ON—O—, ON—N— orON—C-sugars; ON—O—, ON—N— or ON—C— modified or unmodifiedoligonudeotides (comprising at least 5 nucleotides, preferably 5-200nucleotides); ON—O—, ON—N— or ON—C— straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbons; and ON—O—, ON—N— or ON—C— heterocyclic compounds.

Another group of NO adducts for use in the present invention includenitrates that donate, transfer or release nitric oxide, such ascompounds comprising at least one O₂N—O—, O₂N—N—, O₂N—S— or O₂N—C—group. Preferred among these compounds are O₂N—O—, O₂N—N—, O₂N—S— orO₂N—C— polypeptides (the term “polypeptide” includes proteins and alsopolyamino acids that do not possess an ascertained biological function,and derivatives thereof); O₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— amino acids(including natural and synthetic amino acids and their stereoisomers andracemic mixtures); O₂N—O—, O₂N—N—, O₂N—S— or O₂N—C-sugars; O₂N—O—,O₂N—N—, O₂N—S— or O₂N—C— modified and unmodified oligonucleotides(comprising at least 5 nucleotides, preferably 5-200 nucleotides);O₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbons; and O₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— heterocycliccompounds. Preferred examples of compounds comprising at least oneO₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— group include isosorbide dinitrate,isosorbide mononitrate, clonitrate, erythrityltetranitrate, mannitolhexanitrate, nitroglycerin, pentaerythritol-tetranitrate, pentrinitroland propatylnitrate.

Another group of NO adducts are N-oxo-N-nitrosoamines that donate,transfer or release nitric oxide and are represented by the formula:R¹R²—N(O— M⁺)—NO, where R¹ and R² are each independently a polypeptide,an amino acid, a sugar, a modified or unmodified oligonucleotide, astraight or branched, saturated or unsaturated, aliphatic or aromatic,substituted or unsubstituted hydrocarbon, or a heterocylic group, andM³⁰ is as defined herein.

Another group of NO adducts are thionitrates that donate, transfer orrelease nitric oxide and are represented by the formula: R¹—(S)—NO₂,where R¹ is a polypeptide, an amino acid, a sugar, a modified orunmodified oligonucleotide, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbon, or a heterocyclic group. Preferred are those compoundswhere R¹ is a polypeptide or hydrocarbon with a pair or pairs of thiolsthat are sufficiently structurally proximate, i.e., vicinal, that thepair of thiols will be reduced to a disulfide. Compounds which formdisulfide species release nitroxyl ion (NO−) and uncharged nitric oxide(NO·).

The present invention is also directed to compounds that stimulateendogenous NO or elevate levels of endogenous endothelium-derivedrelaxing factor (EDRF) in vivo or are substrates for the enzyme, nitricoxide synthase. Such compounds include, for example, L-arginine,L-homoarginine, and N-hydroxy-L-arginine, including their nitrosated andnitrosylated analogs (e.g., nitrosated L-arginine, nitrosylatedL-arginine, nitrosated N-hydroxy-L-arginine, nitrosylatedN-hydroxy-L-arginine, nitrosated L-homoarginine and nitrosylatedL-homoarginine), precursors of L-arginine and/or physiologicallyacceptable salts thereof, including, for example, citrulline, ornithine,glutamine, lysine, polypeptides comprising at least one of these aminoacids, inhibitors of the enzyme arginase (e.g., N-hydroxy-L-arginine and2(S)-amino-6-oronohexanoic acid), and the substrates for nitric oxidesynthase, cytokines, adenosin, bradykinin, calreticulin, bisacodyl, andphenolphthalein. EDRF is a vascular relaxing factor secreted by theendothelium, and has been identified as nitric oxide (NO) or a closelyrelated derivative thereof (Palmer et al, Nature, 327:524-526 (1987);Ignarro et al, Proc. Natl. Acad. Sci. USA, 84:9265-9269 (1987)).

Compounds of the present invention which have one or more asymmetriccarbon atoms may exist as the optically pure enantiomers, purediastereomers, mixtures of enantiomers, mixtures of diastereomers,racemic mixtures of enantiomers, diastereomeric racemates or mixtures ofdiastereomeric racemates. It is to be understood that the presentinvention anticipates and includes within its scope all such isomers andmixtures thereof.

Another embodiment of the present invention describes compositionscomprising megakaryocytes, at least one thrombopoiesis stimulatingfactor in combination with at least one nitric oxide donor. Thesecompositions enhance the platelet and/or proplatelet production frommegakaryocytes in culture or in vivo. The thrombopoiesis stimulatingfactor can be any described herein or otherwise known in the art, and ispreferably thrombopoietin, interleukin 3, interleukin 6 or interleukin11, Flt 3, stem cell factors, or mixtures thereof; and is morepreferably thrombopoietin (TPO).

The present invention also provides methods for treating and/orpreventing blood platelet disorders in a patient by administering to thepatient a therapeutically effective amount of at least one nitric oxidedonor in combination with at least one thrombopoiesis stimulatingfactor. This method enhances the growth and production of the patient'sown platelets. The compounds can be administered separately or ascomponents of the same composition. The compounds of the presentinvention can also be administered in combination with other medicationsused for the treatment of platelet disorders. These medications includebut are not limited to corticosteroids (such as, for example,dexamethasone, prednisone including those corticosteroids described inthe art, for example, in Goodman and Gilman, The Pharmacological Basisof Therapeutics (9th Ed.), McGraw-Hill, Inc. (1996), the Merck Index onCD-ROM, Twelfth Edition, Version 12:1, (1996), STN Express, file pharand file registry), and immunoglobulins (such as, for example,immunoglobulin G). The treatment of thrombocytopenia usingcorticosteroids and/or immunoglobulins has been previously described.For example, Dunst et al, Am J. Kidney Dis., 31(1):116-120 (1998)describes the use of corticosteroids for the treatment ofthrombocytopenia; Hocker-Schulz et al, Klin. Padiatr., 209(1):30-35(1997) describes the use of corticosteroids and immunoglobulins for thetreatment of acute and chronic immune thrombocytopenia in childhood;Imbach et al, Lancet, 2(8453):464-468 (1985) conducts a multicenterstudy comparing the effect of immunoglobulin versus corticosteroids forthe treatment of thrombocytopenic purpura (the disclosure of each ofthese are incorporated by reference herein in their entirety). Theability to enhance the production of a patient's own plateletseliminates the need for platelet transfusions, which carry thepossibility of transmission of many blood-born infectious diseases.

There has been an increase in the number of platelet transfusions beingperformed. This increase appears to be due to the advances in medicaltechnology and to greater access to such technologies as cardiac surgeryand bone marrow, heart and liver transplants. Platelet transfusion isalso used to hasten the recovery of the platelet counts in thosepatients whose megakaryocytes have been suppressed by chemotherapy orradiation for malignant diseases.

Another aspect of the present invention provides methods for treatingand/or preventing blood platelet disorders in a patient by administeringa therapeutically effective amount of platelets and/or proplateletsproduced in vitro from megakaryocytes in culture by administration of atleast one nitric oxide donor, and, optionally, at least onethrombopoiesis stimulating factor.

Yet another aspect of the present invention provides methods fordecreasing the platelet counts in a patient by administering aneffective amount of at least one compound that inhibits the productionof the patient's own nitric oxide. Such compounds include inhibitors ofnitric oxide synthase (NOS) such as, for example,N^(G)-nitro-L-arginine, N^(G)-amino-L-arginine,N^(G)—N^(G)-dmethyl-arginine and N^(G)-mono-methyl-L-arginine, and thelike.

For producing platelets and/or proplatelets in vitro, an effectiveamount of at least one nitric oxide donor (i.e., at least one compoundthat donates, transfers, or releases nitric oxide, or induces theproduction of endogenous nitric oxide or endothelium-derived relaxingfactor or is a substrate for nitric oxide synthase or a pharmaceuticallyacceptable salt thereof) is added to at least one megakaryocyte. Inother in vitro embodiments, an effective amount of at least one nitricoxide donor and at least one thrombopoiesis stimulating factor are addedto the at least one megakaryocyte, where the at least one nitric oxidedonor and the at least one thrombopoiesis stimulating factor are addedseparately to the at least one megakaryocyte or are in the form of acomposition when they are added to the at least one megakaryocyte. Inother alternative in vitro embodiments, the at least one nitric oxidedonor can be added to the at least one megakaryocyte simultaneouslywith, subsequent to, or prior to adding the at least one thrombopoiesisstimulating factor to the at least one megakaryocyte. In preferred invitro embodiments for producing platelets and/or proplatelets, at leastone thrombopoiesis stimulating factor is added to at least onemegakaryocyte, and subsequently at least one nitric oxide donor is addedto the at least one megakaryocyte and the at least one thrombopoiesisstimulating factor. As one skilled in the art will recognize from thedescription herein, after the platelets and/or proplatelets are producedin vitro following the methods described herein, they can then beadministered in a therapeutically effective amount to a patient to treator prevent a blood platelet disorder.

When administered in vivo, the compositions of the present invention canbe administered alone or in combination with pharmaceutically acceptablecarriers or diluents and in dosages described herein using methods thatallow access to the patient's blood stream and that allow contact withthe patient's megakaryocytes. When at least one thrombopoiesisstimulating factor and at least one nitric oxide donor are administeredtogether in the form of a composition, they can also be used incombination with one or more additional compounds. Alternatively, thenitric oxide donor(s) can be administered simultaneously with,subsequently to, or prior to administration of the thrombopoiesisstimulating factor(s) and/or other additional compound(s). The preferredmethod of administration of the nitric oxide donor is subsequent to theadministration of the thrombopoiesis stimulating factor.

The dosage regimen for treating and/or preventing blood plateletdisorders with the compounds and/or compositions of the presentinvention is selected in accordance with a variety of factors, includingthe age, weight, sex, diet and medical condition of the patient, theseverity of the disease, the route of administration, pharmacologicalconsiderations such as the activity, efficacy, pharmacokinetic andtoxicology profiles of the particular compound used, whether a drugdelivery system is used and whether the compound is administered as partof a drug combination. Thus, the dosage regimen actually used can varywidely and therefore can deviate from the preferred dosage regimen setforth herein.

The compounds and compositions of the present invention can beadministered by any available and effective delivery system including,but not limited to, orally, bucally, parenterally, by inhalation spray,by topical application, by injection, by transurethral drug delivery,transdermally, or rectally (e.g., by the use of suppositories) in dosageunit formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles, as desired. Parenteralincludes subcutaneous injections, intravenous injections, intramuscularinjections, intrasternal injections, and infusion techniques. Parenteralalso includes injection, which can be conducted using any effectiveinjection system including, but not limited to, conventionalsyringe-and-needle systems or needleless injection devices.

Transdermal drug administration, which is known to one skilled in theart, involves the delivery of pharmaceutical agents via percutaneouspassage of the drug into the systemic circulation of the patient.Topical administration, which is well known to one skilled in the art,involves the delivery of pharmaceutical agents via percutaneous passageof the drug into the systemic circulation of the patient. Topicaladministration can also involve transdermal patches or iontophoresisdevices. Other components can be incorporated into the transdermalpatches as well. For example, compositions and/or transdermal patchescan be formulated with one or more preservatives or bacteriostaticagents including, but not limited to, methyl hydroxybenzoate, propylhydroxybenzoate, chlorocresol, benzalkonium chloride, and the like.

Dosage forms for topical administration of the compounds andcompositions of the present invention preferably include creams, sprays,lotions, gels, ointments, emulsions, liposomes, foams, and the like.Administration of the cream, spray, lotion, gel, ointment, emulsion,coating, liposome, or foam can be accompanied by the use of anapplicator for drug delivery using a syringe with or without a needle,and is within the skill of the art.

Solid dosage forms for oral administration can include capsules,tablets, effervescent tablets, chewable tablets, pills, powders,effervescent powders, sachets, granules and gels. In such solid dosageforms, the active compounds can be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms can alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate. In thecase of capsules, tablets, effervescent tablets, and pills, the dosageforms can also comprise buffering agents. Soft gelatin capsules can beprepared to contain a mixture of the active compounds or compositions ofthe present invention and vegetable oil. Hard gelatin capsules cancontain granules of the active compound in combination with a solid,pulverulent carrier such as lactose, saccharose, sorbitol, mannitol,potato starch, corn starch, amylopectin, cellulose derivatives ofgelatin. Tablets and pills can be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

Suppositories for administration of the compounds and compositions ofthe invention can be prepared by mixing the compounds or compositionswith a suitable nonirritating excipient such as cocoa butter andpolyethylene glycols which are solid at room temperature but liquid atbody temperature, such that they will melt and release the drug.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents and/or suspendingagents. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be used are water,Ringer's solution, and isotonic sodium chloride solution. Sterile fixedoils are also conventionally used as a solvent or suspending medium.

The compounds and compositions of the present invention will typicallybe administered in a pharmaceutical composition containing one or morecarriers or excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral application whichdo not deleteriously react with the active compounds. Examples ofpharmaceutically acceptable carriers include, for example, water, saltsolutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils,polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,lactose, amylose, magnesium stearate, talc, surfactants, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, and the like. The compositions can also includeone or more permeation enhancers including, for example,dimethylsulfoxide (DMSO), dimethyl formamide (DMF),N,N-dimethylacetamide (DMA), decylmethylsulfoxide (ClOMSO), polyethyleneglycol monolaurate (PEGML), glyceral monolaurate, lecithin,1-substituted azacycloheptan-2-ones, particularly1-N-dodecylcyclaza-cylcoheptan-2-ones (available under the trademarkAZONE™ from Nelson Research & Development Co., Irvine, Calif.), alcoholsand the like.

The pharmaceutical preparations can be sterilized and if desired, rnrxedwith auxiliary agents which do not deleteriously react with the activecompounds, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavoring and/or aromatic substances, and the like. For parenteralapplication, particularly suitable vehicles consist of solutions,preferably oily or aqueous solutions, as well as suspensions, emulsions,or implants. Aqueous suspensions may contain substances which increasethe viscosity of the suspension and include, for example, sodiumcarboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers.

The composition, if desired, can also contain minor amounts of wettingagents, emulsifying agents and/or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulations can include standard carriers suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike.

Various delivery systems are known and can be used to administer thecompounds or compositions of the present invention, including, forexample, encapsulation in liposomes, microbubbles, emulsions,micropartides, microcapsules and the like. The required dosage can beadministered as a single unit or in a sustained release form.

The bioavailability of the compositions can be enhanced by micronizationof the formulations using conventional techniques such as grinding,milling, spray drying and the like in the presence of suitableexcipients or agents such as phospholipids or surfactants.

The compounds and compositions of the present invention can beformulated as pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts include, for example, alkali metal salts and additionsalts of free acids or free bases. The nature of the salt is notcritical, provided that it is pharmaceutically-acceptable. Suitablepharmaceutically-acceptable acid addition salts may be prepared from aninorganic acid or from an organic acid. Examples of such inorganic acidsinclude, but are not limited to, hydrochloric, hydrobrornic, hydroiodic,nitric (nitrate salt), nitrous (nitrite salt), carbonic, sulfuric andphosphoric acid and the like. Appropriate organic acids include, but arenot limited to, aliphatic, cycloaliphatic, aromatic, heterocyclic,carboxylic and sulfonic classes of organic acids, such as, for example,formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,toluenesulfonic, 2-hydroxyethane-sulfonic, sulfanilic, stearic, algenic,hydroxy-butyric, cyclohexylaminosulfonic, galactaric and galacturonicacid and the like. Suitable pharmaceutically-acceptable base additionsalts include, but are not limited to, metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium and zinc ororganic salts made from primary, secondary and tertiary amines, cyclicamines, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine and the like. All of these salts may be prepared byconventional means from the corresponding compound by reacting, forexample, the appropriate acid or base with the compound.

“Therapeutically effective amount” refers to the amount of the nitricoxide donor and/or thrombopoiesis stimulating factor which is effectiveto achieve its intended purpose by allowing access into a patient'sblood stream and enable contact with the patient's own megakaryocytes.“Therapeutically effective amount” also refers to the amount ofplatelets and/or proplatelets necessary to prevent or treat a blooddisorder as described herein. While individual patient needs may vary,determination of optimal ranges for effective amounts of each nitricoxide adduct is within the skill of the art. Generally the dosageregimen for treating a condition with the compounds and/or compositionsof this invention is selected in accordance with a variety of factors,including the type, age, weight, sex, diet and medical condition of thepatient, the severity of the dysfunction, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetic and toxicology profiles of the particular compound used,whether a drug delivery system is used, and whether the compound isadministered as part of a drug combination and can be adjusted by oneskilled in the art. Thus, the dosage regimen actually employed may varywidely and therefore may deviate from the preferred dosage regimen setforth herein.

A particularly preferred method of administering the thrombopoiesisstimulating factor and the nitric oxide donor is intravenously. Ifdesired the compounds and/or compositions can be administeredsubcutaneously. When systemically administered, the therapeuticcompositions are in the form of pyrogen-free, parenterally acceptableaqueous solutions.

The present invention also provides pharmaceutical kits comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compounds and/or compositions of the present invention,including, at least, one or more thrombopoiesis stimulating factors incombination with one or more of the NO donors described herein. Suchkits can also include, for example, other compounds and/or compositions(e.g., antibiotics), a device(s) for administering the compounds and/orcompositions, and written instructions in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which instructions can alsoreflects approval by the agency of manufacture, use or sale for humanadministration.

EXAMPLES

The invention is further demonstrated in the following examples. Theexamples are for purposes of illustration only, and are not intended tolimit the scope of the present invention or claims.

As described in Example 1 herein, treatment of megakaryocyte cell line,Meg-01 cells, with the thrombopoiesis stimulating factor TPO, followedby the nitric oxide donor S-nitroso-glutathione (SNO-glu), promotedplatelet-like particle formation by the megakaryocytes relative to thecells that were not treated with SNO-glu. The increase in the productionof the platelet-like particles was determined by flow cytometry. Theplatelet-like particles were found to be functional, to expressactivated glycoprotein lIb/IIIa on their surface in response tothrombin-receptor activating peptide, and to form aggregates whenactivated in the presence of calcium and fibrinogen. As described inExample 2, these cells were shown to produce CGMP thereby suggestingthat nitric oxide might be involved in the process as it is known thatcGMP is elevated by nitric oxide. Additionally mice in which the iNOSgene has been deleted have one-half the platelet counts of wild typemice, supporting the importance of endogenous nitric oxide inthrombopoiesis (Table 2).

Example 1 Platelet Production from Megakaryocytes

The production of platelet-sized particles in the Meg-01 cell line usingflow cytometry was determined. Human megakaryoblastic leukemia cell lineMeg-01 was obtained from American Type Culture Collection (Manassas,Va.) and grown in phenol red-free RPMI-1640 media (Gibco-BRL, GrandIsland, N.Y.) supplemented with 10% fetal bovine serum at 37° C. in ahumidified atmosphere of 5% CO₂. The experiment to monitor plateletproduction by the megakaryocytoid cell line, Meg-01, were performed asfollows: 10 ml of Meg-01 cells (1×10⁵/ml) were treated with 100 ng/ml ofthrombopoietin (TPO, Amgen, Thousand Oaks, Calif.) for 72 hours. Thecells were then centrifuged at 1000 rpm for 10 minutes, washed withphosphate buffered saline (PBS) and resuspended in RPMI-1640 media.Meg-01 cells, in the presence and absence of TPO, were treated with 100μM SNO-glu for 2 hours. The cells were collected by centrifugation (1000rpm for 10 minutes), washed once in PBS, resuspended in 500 μl of 3%formaldehyde, and allowed to fix for 20 minutes at room temperature.After fixation, 100 μl of cells were labeled with fluoresceinisothiocyanate-conjugated (FITC)-conjugated monoclonal mouse anti-humanplatelet glycoprotein IIIa, CD61 (GPIIIa, DAKO Corp. Carpinteria,Calif.) at a near saturating concentration and incubated at roomtemperature for 20 minutes. The sample was diluted in 100 μl of PBS andanalyzed on a Coulter EPICS XL flow cytometer (Coulter, Miami, Fla.).DNA check flow cytometry beads purchased from Coulter were used fordaily instrument calibration. Appropriate color compensation was set forFITC fluorescence and phycoerythrin fluorescence using 525 nm and 575 nmband pass filters, respectively. All data were saved in flow cytometrylist mode files and analyzed using Coulter ELITE software, version 2.21.Platelet-sized particles were identified by characteristic log forwardlight scatter (LFS). Only those particles that bound theanti-GPIIIa-FITC antibody (CD41 FITC-conjugated antibody) (Dako,Denmark) were analyzed (by gating the fluorescence signal).

FIG. 1A is the flow cytometric analysis of untreated, cultured samplesof Meg-01 cells positive for GPIIIa.

FIG. 1B shows that Meg-01 cells treated with 100 μMS-nitroso-glutathione (SNO-glu) show the formation of GPIIIa positiveplatelet-sized particle population in addition to GPIIIa positive Meg-01cells.

FIG. 1C shows an enhanced production of GPIIIa positive platelet-sizedparticles by the flow cytometric analysis of the Meg-01 cells pretreatedwith 100 ng/ml thrombopoietin, followed by treatment with 100 μMSNO-glu.

For experiments in which potential platelet aggregation was monitored,the cell suspension was centrifuged at 150 g to collect a platelet-richfraction. The platelet-rich fraction was incubated with 10 μMthrombin-receptor activating protein (TRAP, Bachem, Torrance, Calif.),2.5 mM CaCl₂, and 600 μg/ml fibrinogen (Chromogenix, Sweden). Thepotential platelet aggregates were then treated as described above foranalysis by flow cytometry. FIG. 1D shows a shift in size of theplatelet population which is suggestive of aggregate formation.

Example 2 Production of cGMP from Megakaryocytes treated with SNO-glu

The Meg-01 cells were grown as described in Example 1. The Med-01 cells,in the presence and absence of TPO, were treated with 100 μM SNO-glu forvarying lengths of time. The cells were collected by centrifugation(1000 rpm for 10 minutes), and then lysed with 6% trichloroacteic acid(TCA). The cGMP was extracted with distilled water-saturated ether. ThecGMP was quantified using the standard ELISA assay (Caymen ChemicalInc., Ann Arbor, Mich.). The results summarized in Table 1 show that theaddition of SNO-glu results in an increased production of cGMP. Thissuggests that nitric oxide mediates platelet production. Interestingly,the addition of TPO during the SNO-glu treatment completely suppressedthe production of cGMP.

TABLE 1 Treatment Group pmol cGMP/mg Protein (n = 2) Untreated 1.3 100μM SNO-Glu, 5 minutes 263.1 100 μM SNO-Glu, 10 minutes 461.2 100 μMSNO-Glu, 20 minutes 472.9 100 μM SNO-Glu, 30 minutes 611.3 100 μMSNO-Glu in the 0.960 presence of TPO, 30 minutes

Example 3 Platelet Counts from iNOS(−/−) and eNOS (−/−) mice

Blood was collected from age-matched wild type (+/+), iNOS(−/−) and eNOS(−/−) mice. The blood was withdrawn from the aorta of the euthanizedmice into a syringe containing 10% trisodium citrate (90 mM) to preventcoagulation and carefully placed in a tube (17 mm×199 mm) followed bycentrifugation at 190 g to prepare the platelet-rich plasma. Theplatelets were counted using a Coulter counter, model Z (Coulter Inc.,Haileah, Fla.). As summarized in Table 2, in 17-week-old knockout mice,there was a 50% decrease in platelet count in INOS (−/−) in comparisonto strain- and age-matched control animals and eNOS (−/−) animals: theiNOS knockout mice (−/−) had an average platelet count of 320,000/ 1while the wild type animals had an average platelet count of 569,000/ 1,a difference which was highly statistically significant (p=0.00325). Nodifference was noted between the platelet counts of the eNOS (−/−) miceand the wild type mice (564,000/ 1). These data suggest that endogenousnitric oxide is involved in thrombopoiesis.

TABLE 2 Group Platelet Count (/μl) Number of Animals Wild Type (+/+)569,000 ± 400 7 iNOS (−/−) 320,000 ± 700 10 eNOS (−/−) 540,000 ± 600 3

The disclosure of each patent, patent application and publication citedor described herein is hereby incorporated by reference herein in itsentirety.

Although the invention has been set forth in detail, one skilled in theart will appreciate that numerous changes and modifications can be madeto the invention without departing from the spirit and scope thereof.

What is claimed is:
 1. A composition comprising at least onemegakaryocyte, at least one thrombopoeisis stimulating factor and atleast one compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor or is a substrate for nitric oxide synthase or apharmaceutically acceptable salt thereof.
 2. The composition of claim 1,wherein the compound that donates, transfers, or releases nitric oxide,or induces the production of endogenous nitric oxide orendothelium-derived relaxing factor or is a substrate for nitric oxidesynthase is an S-nitrosothiol.
 3. The composition of claim 2, whereinthe S-nitrosothiol is S-nitroso-N-acetylcysteine, S-nitroso-captopril,S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine,S-nitroso-cysteine or S-nitroso-glutathione.
 4. The composition of claim3, wherein the S-nitrosothiol is S-nitroso-glutathione.
 5. Thecomposition of claim 2, wherein the S-nitrosothiol is: (i)HS(C(R_(e))(R_(f)))_(m)SNO; (ii) ONS(C(R_(e))(R_(f)))_(m)R_(e); and(iii) H₂N—CH(CO₂H)—(CH₂)_(m)—C(O)NH—CH(CH₂SNO)—C(O)NH—CH₂—CO₂H; whereinm is an integer from 2 to 20; R_(e) and R_(f) are each independently ahydrogen, an alkyl, a cycloalkoxy, a halogen, a hydroxy, anhydroxyalkyl, an alkoxyalkyl, an arylheterocyclic ring, an alkylaryl, acycloalkylalkyl, a heterocyclicalkyl, an alkoxy, a haloalkoxy, an amino,an alkylamino, a dialkylamino, an arylamino, a diarylamino, analkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a sulfonic acid, asulfonic ester, an alkylsulfonic acid, an arylsulfonic acid, anarylalkoxy, an alkylthio, an arylthio, a cycloalkylthio, a cycloalkenyl,a cyano, an aminoalkyl, an aminoaryl, an aryl, an arylalkyl, analkylaryl, a carboxamido, a alkylcarboxamido, an arylcarboxamido, anamidyl, a carboxyl, a carbamoyl, a carbamate, an alkylcarboxylic acid,an arylcarboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, acarboxylic ester, an alkylcarboxylic ester, an arylcarboxylic ester, ahaloalkoxy, a sulfonamido, an alkylsulfonamido, an arylsulfonamido, asulfonic ester, a urea, a phosphoryl, a nitro, —T—Q , or(C(R_(e))(R_(f)))_(k)—T—Q, or R_(e) and R_(f) taken together with thecarbons to which they are attached form a carbonyl, a methanthial, aheterocyclic ring, a cycloalkyl group or a bridged cycloalkyl group; Qis —NO or —NO₂; and T is independently a covalent bond, a carbonyl, anoxygen, —S(O)_(o)— or —N(R_(a))R_(i)—, wherein o is an integer from 0 to2, R_(a) is a lone pair of electrons, a hydrogen or an alkyl group;R_(i) is a hydrogen, an alkyl, an aryl, an alkylcarboxylic acid, an arylcarboxylic acid, an alkylcarboxylic ester, an arylcarboxylic ester, analkylcarboxamido, an arylcarboxamido, an alkylaryl, an alkylsulfinyl, analkylsulfonyl, an arylsulfinyl, an arylsulfonyl, a sulfonamido, acarboxamido, a carboxylic ester, an amino alkyl, an amino aryl,—CH₂—C(T—Q)(R_(e))(R_(f)), or —(N₂O₂−)⁻·M⁺, wherein M⁺ is an organic orinorganic cation; with the proviso that when R^(i) is—CH₂—C(T—Q)(R_(e))(R_(f)) or —(N₂O₂−)·M⁺; then “—T—Q” can be a hydrogen,an alkyl group, an alkoxyalkyl group, an aminoalkyl group, a hydroxygroup or an aryl group.
 6. The composition of claim 1, wherein the atleast one compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor, or is a substrate for nitric oxide synthase is: (i) acompound that comprises at least one ON—O—, ON—N— or ON—C— group; (ii) acompound that comprises at least one O₂N—O—, O₂N—N—, O₂N—S— or —O₂N—C—group; (iii) a N-oxo-N-nitrosoamine having the formula: R¹R²—N(O—M⁺)—NO,wherein R¹ and R² are each independently a polypeptide, an amino acid, asugar, an oligonucleotide, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbon, or a heterocyclic group, and M⁺ is an organic or inorganiccation.
 7. The composition of claim 6, wherein the compound comprisingat least one ON—O—, ON—N— or ON—C— group is an ON—O-polypeptide, anON—N-polypeptide, an ON—C-polypeptide, an ON—O-amino acid, an ON—N-aminoacid, an ON—C-amino acid, an ON—O-sugar, an ON—N-sugar, an ON—C-sugar,an ON—O-oligonucleotide, an ON—N-oligonucleotide, anON—C-oligonucleotide, a straight or branched, saturated or unsaturated,substituted or unsubstituted, aliphatic or aromatic ON—O-hydrocarbon, astraight or branched, saturated or unsaturated, substituted orunsubstituted, aliphatic or aromatic ON—N-hydrocarbon, a straight orbranched, saturated or unsaturated, substituted or unsubstituted,aliphatic or aromatic ON—C-hydrocarbon, an ON—O-heterocyclic compound,an ON—N-heterocyclic compound or a ON—C-heterocyclic compound.
 8. Thecomposition of claim 6, wherein compound comprising at least one O₂N—O—,O₂N—N—, O₂N—S— or O₂N—C— group is an O₂N—O-polypeptide, anO₂N—N-polypeptide, an O₂N—S-polypeptide, an O₂N—C-polypeptide, anO₂N—O-amiino acid, O₂N—N-amino acid, O₂N—S-amino acid, an O₂N—C-aminoacid, an O₂N—O-sugar, an O₂N—N-sugar, O₂N—S-sugar, an O₂N—C-sugar, anO₂N—O -oligonucleotide, an O₂N—N-oligonucleotide, anO₂N—S-oligonucleotide, an O₂N—C-oligonucleotide, a straight or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted O₂N—O-hydrocarbon, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedO₂N—N-hydrocarbon, a straight or branched, saturated or unsaturated,aliphatic or aromatic, substituted or unsubstituted O₂N—S-hydrocarbon, astraight or branched, saturated or unsaturated, aliphatic or aromatic,substituted or unsubstituted O₂N—C-hydrocarbon, an O₂N—O-heterocycliccompound, an O₂N—N-heterocyclic compound, an O₂N—S-heterocyclic compoundor an O₂N—C-heterocyclic compound.
 9. The composition of claim 1,wherein the at least one compound that donates, transfers, or releasesnitric oxide, or induces the production of endogenous nitric oxide orendothelium-derived relaxing factor, or is a substrate for nitric oxidesynthase, is L-arginine, L-homoarginine, N-hydroxy-L-arginine,nitrosated L-arginine, nitrosylated L-arginine, nitrosatedN-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, citrulline,ornithine, glutamine, lysine, polypeptides comprising at least one ofthese amino acids or inhibitors of the enzyme arginase, cytokines,adenosin, bradykinin, calreticulin, bisacodyl, and phenolphthalein. 10.The composition of claim 1, wherein the thrombopoeisis stimulatingfactor is interleukin 1, interleukin 2, interleukin 3, interleukin 4,interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin9, interleukin 10, interleukin 11, interleukin 12, interleukin 13,interleukin 14, interleukin 15, erythropoietin, thrombopoietin, stemcell factor, flt-3 ligand, granulocyte colony stimulating factor,granulocyte macrophage colony stimulating factor, tumor growth factorbeta, tumor necrosis factor alpha, interferon, fibroblast growth factor,platelet-derived growth factor, insulin-like growth factors, leukemiainhibitor factor, megakaryocyte colony stimulating factor, or mixturesthereof.
 11. The composition of claim 10, wherein the thrombopoeisisstimulating factor is thrombopoietin.
 12. The composition of claim 1,wherein the at least one megakaryocyte is in culture.
 13. Thecomposition of claim 12, wherein the at least one megakaryocyte inculture is obtained from a cell line, a stem cell, a tissue, bonemarrow, peripheral blood, a liver, a fetal liver or a patient's ownblood megakaryocytic precursors.
 14. A method for producing plateletsand/or proplatelets in vitro comprising adding the composition of claim1 to at least one megakaryocyte in culture.
 15. The method of claim 14,wherein the at least one megakaryocyte in culture is obtained from acell line, a stem cell, a tissue, bone marrow, peripheral blood, aliver, a fetal liver or a patient's own blood megakaryocytic precursors.16. A method for producing platelets and/or proplatelets in vivo in apatient in need thereof comprising administering to the patient aneffective amount of the composition of claim
 1. 17. A method fortreating or preventing a blood platelet disorder in a patient in needthereof comprising administering to the patient a therapeuticallyeffective amount of the composition of claim
 1. 18. A method forproducing platelets and/or proplatelets comprising: providing at leastone megakaryocyte; and adding an effective amount of at least onethrombopoeisis stimulating factor and at least one compound thatdonates, transfers, or releases nitric oxide, or induces the productionof endogenous nitric oxide or endothelium-derived relaxing factor or isa substrate for nitric oxide synthase or a pharmaceutically acceptablesalt thereof to the at least one megakaryocyte to produce plateletsand/or proplatelets.
 19. The method of claim 18, wherein themegakaryocyte is in culture.
 20. The method of claim 19, wherein the atleast one megakaryocyte in culture is obtained from a cell line, a stemcell, a tissue, bone marrow, peripheral blood, a liver, a fetal liver ora patient's own blood megakaryocytic precursors.
 21. The method of claim18, wherein the megakaryocyte is in vivo.
 22. The method of claim 18,wherein the thrombopoeisis stimulating factor is added to the at leastone megakaryocyte prior to the step of adding the compound that donates,transfers, or releases nitric oxide, or induces the production ofendogenous nitric oxide or endothelium-derived relaxing factor or is asubstrate for nitric oxide synthase.
 23. The method of claim 18, whereinthe thrombopoeisis stimulating factor is interleukin 1, interleukin 2,interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin7, interleukin 8, interleukin 9, interleukin 10, interleukin 11,interleukin 12, interleukin 13, interleukin 14, interleukin 15,erythropoietin, thrombopoietin, stem cell factor,flt-3 ligand,granulocyte colony stimulating factor, granulocyte macrophage colonystimulating factor, tumor growth factor beta, tumor necrosis factoralpha, interferon, fibroblast growth factor, platelet-derived growthfactor, insulin-like growth factors, leukemia inhibitor factor ormegakaryocyte colony stimulating factor, or a mixture thereof.
 24. Themethod of claim 23, wherein the thrombopoeisis stimulating factor isthrombopoietin.
 25. The method of claim 18, wherein the compound thatdonates, transfers, or releases nitric oxide, or induces the productionof endogenous nitric oxide or endothelium-derived relaxing factor or isa substrate for nitric oxide synthase is an S-nitrosothiol.
 26. Themethod of claim 25, wherein the S-nitrosothiol isS-nitroso-N-acetylcysteine, S-nitroso-captopril,S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine,S-nitroso-cysteine or S-nitroso-glutathione.
 27. The method of claim 26,wherein the S-nitrosothiol is S-nitroso-glutathione.
 28. The method ofclaim 25, wherein the S-nitrosothiol is: (i) HS(C(R_(e))(R_(f)))_(m)SNO;(ii) ONS(C(R_(e))(R_(f)))_(m)R_(e); and (iii)H₂N—CH(CO₂H)—(CH₂)_(m)—C(O)NH—CH(CH₂SNO)—C(O)NH—CH₂—CO₂H; wherein m isan integer from 2 to 20; R_(e) and R_(f) are each independently ahydrogen, an alkyl, a cycloalkoxy, a halogen, a hydroxy, anhydroxyalkyl, an alkoxyalkyl, an arylheterocyclic ring, an alkylaryl, acycloalkylalkyl, a heterocyclicalkyl, an alkoxy, a haloalkoxy, an amino,an alkylamino, a dialkylamino, an arylamino, a diarylamino, analkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a sulfonic acid, asulfonic ester, an alkylsulfonic acid, an arylsulfonic acid, anarylalkoxy, an alkylthio, an arylthio, a cycloalkylthio, a cycloalkenyl,a cyano, an aminoalkyl, an aminoaryl, an aryl, an arylalkyl, analkylaryl, a carboxamido, a alkylcarboxamido, an arylcarboxamido, anamidyl, a carboxyl, a carbamoyl, a carbamate, an alkylcarboxylic acid,an arylcarboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, acarboxylic ester, an alkylcarboxylic ester, an arylcarboxylic ester, ahaloalkoxy, a sulfonamido, an alkylsulfonamido, an arylsulfonamido, asulfonic ester, a urea, a phosphoryl, a nitro, —T—Q , or(C(R_(e))(R_(f)))_(k)—T—Q, or R_(e) and R_(f) taken together with thecarbons to which they are attached form a carbonyl, a methanthial, aheterocyclic rinrg, a cycloalkyl group or a bridged cycloalkyl group; Qis —NO or —NO₂; and T is independently a covalent bond, a carbonyl, anoxygen, —S(O)_(o)— or —N(R_(a))R_(i)—, wherein o is an integer from 0 to2, R_(a) is a lone pair of electrons, a hydrogen or an alkyl group;R_(i) is a hydrogen, an alkyl, an aryl, an alkylcarboxylic acid, an arylcarboxylic acid, an alkylcarboxylic ester, an arylcarboxylic ester, analkylcarboxamido, an arylcarboxamido, an alkylaryl, an alkylsulfinyl, analkylsulfonyl, an arylsulfinyl, an arylsulfonyl, a sulfonamido, acarboxamido, a carboxylic ester, an amino alkyl, an amino aryl,—CH₂—C(T—Q)(R_(e))(R_(f)), or —(N₂O₂—)⁻·M⁺, wherein M⁺ is an organic orinorganic cation; with the proviso that when R_(i) is—CH₂—C(T—Q)(R_(e))(R_(f)) or —(N₂O₂—)·M⁺; then “—T—Q” can be a hydrogen,an alkyl group, an alkoxyalkyl group, an aminoalkyl group, a hydroxygroup or an aryl group.
 29. The method of claim 18, wherein the at leastone compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor, or is a substrate for nitric oxide synthase is: (i) acompound that comprises at least one ON—O—, ON—N— or ON—C— group; (ii) acompound that comprises at least one O₂N—O—, O₂N—N—, O₂N—S— or —O₂N—C—group; (iii) a N-oxo-N-nitrosoamine having the formula: R¹R²—N(O—M⁺)—NO,wherein R¹ and R² are each independently a polypeptide, an amino acid, asugar, an oligonucleotide, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbon, or a heterocyclic group, and M⁺ is an organic or inorganiccation.
 30. The method of claim 29, wherein the compound comprising atleast one ON—O—, ON—N— or ON—C— group is an ON—O-polypeptide, anON—N-polypeptide, an ON—C-polypeptide, an ON—O-amino acid, an ON—N-aminoacid, an ON—C-amino acid, an ON—O-sugar, an ON—N-sugar, an ON—C-sugar,an ON—O-oligonucleotide, an ON—N-oligonucleotide, anON—C-oligonucleotide, a straight or branched, saturated or unsaturated,substituted or unsubstituted, aliphatic or aromatic ON—O-hydrocarbon, astraight or branched, saturated or unsaturated, substituted orunsubstituted, aliphatic or aromatic ON—N-hydrocarbon, a straight orbranched, saturated or unsaturated, substituted or unsubstituted,aliphatic or aromatics ON—C-hydrocarbon, an ON—O-heterocyclic compound,an ON—N-heterocyclic compound or a ON—C-heterocyclic compound.
 31. Themethod of claim 29, wherein compound comprising at least one O₂N—O—,O₂N—N—, O₂N—S— or O₂N—C— group is an O₂N—O-polypeptide, anO₂N—N-polypeptide, an O₂N—S-polypeptide, an O₂N—C-polypeptide, anO₂N—O-amino acid, O₂N—N-amino acid, O₂N—S-amino acid, an O₂N—C-aminoacid, an O₂N—O-sugar, an O₂N—N-sugar, O₂N—S-sugar, an O₂N—C-sugar, anO₂N—O-oligonucleotide, an O₂N—N-oligonucleotide, anO₂N—S-oligonucleotide, an O₂N—C-oligonucleotide, a straight or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted O₂N—O-hydrocarbon, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedO₂N—N-hydrocarbon, a straight or branched, saturated or unsaturated,aliphatic or aromatic, substituted or unsubstituted O₂N—S-hydrocarbon, astraight or branched, saturated or unsaturated, aliphatic or aromatic,substituted or unsubstituted O₂N—C-hydrocarbon, an O₂N—O-heterocycliccompound, an O₂N—N-heterocyclic compound, an O₂N-S-heterocyclic compoundor an O₂N-C-heterocyclic compound.
 32. The method of claim 18, whereinthe at least one compound that donates, transfers, or releases nitricoxide, or induces the production of endogenous nitric oxideorendothelium-derived relaxing factor, or is a substrate for nitric oxidesynthase, is L-arginine, L-homoarginine, N-hydroxy-L-arginine,nitrosated L-arginine, nitrosylated L-arginine, nitrosatedN-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, citrulline,ornithine, glutamine, lysine, polypeptides comprising at least one ofthese amino acids or inhibitors of the enzyme arginase, cytokines,adenosin, bradykinin, calreticulin, bisacodyl, and phenolphthalein. 33.The method of claim 18, further comprising adding at least onecorticosteroid and/or at least one immunoglobulin.
 34. The method ofclaim 33, wherein the corticosteroid is dexamethasone or prednisone. 35.The method of claim 33, wherein the immunoglobulin is immunoglobulin G.36. A method for decreasing platelet counts in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of at least one compound that inhibits the production of thepatient's nitric oxide synthesis, wherein the compound that inhibits theproduction of the patient's nitric oxide synthesis isN^(G)-nitro-L-arginine, N^(G)-amino-L-arginine,N^(G)—N^(G)-dimethylarginine or N^(G)-mono-methyl-L-arginine, ormixtures thereof.
 37. A composition comprising at least onethrombopoeisis stimulating factor and at least one compound thatdonates, transfers, or releases nitric oxide, or induces the productionof endogenous nitric oxide or endothelium-derived relaxing factor or isa substrate for nitric oxide synthase.
 38. The composition of claim 37,wherein the thrombopoeisis stimulating factor is interleukin 1,interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin6, interleukin 7, interleukin 8, interleukin 9, interleukin 10,interleukin 11, interleukin 12, interleukin 13, interleukin 14,interleukin 15, erythropoietin, thrombopoietin, stem cell factor, flt-3ligand, granulocyte colony stimulating factor, granulocyte macrophagecolony stimulating factor, tumor growth factor beta, tumor necrosisfactor alpha, interferon, fibroblast growth factor, platelet-derivedgrowth factor, insulin-like growth factors, leukemia inhibitor factor ormegakaryocyte colony stimulating factor, or mixtures thereof.
 39. Thecomposition of claim 38, wherein the thrombopoeisis stimulating factoris thrombopoietin.
 40. The composition of claim 37, wherein the compoundthat donates, transfers, or releases nitric oxide, or induces theproduction of endogenous nitric oxide or endothelium-derived relaxingfactor or is a substrate for nitric oxide synthase is an S-nitrosothiol.41. The composition of claim 40, wherein the S-nitrosothiol isS-nitroso-N-acetylcysteine, S-nitroso-captopril,S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine,S-nitroso-cysteine or S-nitroso-glutathione.
 42. The composition ofclaim 41, wherein the S-nitrosothiol is S-nitroso-glutathione.
 43. Thecomposition of claim 40, wherein the S-nitrosothiol is: (i)HS(C(R_(e))(R_(f)))_(m)SNO; (ii) ONS(C(R_(e))(R_(f)))_(m)R_(e); and(iii) H₂N—CH(CO₂H)—(CH₂)_(m)—C(O)NH—CH(CH₂SNO)—C(O)NH—CH₂—CO₂H; whereinm is an integer from 2 to 20; R_(e) and R_(f) are each independently ahydrogen, an alkyl, a cycloalkoxy, a halogen, a hydroxy, anhydroxyalkyl, an alkoxyalkyl, an arylheterocyclic ring, an alkylaryl, acycloalkylalkyl, a heterocyclicalkyl, an alkoxy, a haloalkoxy, an amino,an alkylamino, a dialkylamino, an arylamino, a diarylamino, analkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a sulfonic acid, asulfonic ester, an alkylsulfonic acid, an arylsulfdnic acid, anarylalkoxy, an alkylthio, an arylthio, a cycloalkylthio, a cycloalkenyl,a cyano, an aminoalkyl, an aminoaryl, an aryl, an arylalkyl, analkylaryl, a carboxamido, a alkylcarboxamido, an arylcarboxamido, anamidyl, a carboxyl, a carbamoyl, a carbamate, an alkylcarboxylic acid,an arylcarboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, acarboxylic ester, an alkylcarboxylic ester, an arylcarboxylic ester, ahaloalkoxy, a sulfonamido, an alkylsulfonamido, an arylsulfonamido, asulfonic ester, a urea, a phosphoryl, a nitro, —T—Q , or(C(R_(e))(R_(f)))_(k)—T—Q, or R_(e) and R_(f) taken together with thecarbons to which they are attached form a carbonyl, a methanthial, aheterocyclic ring, a cycloalkyl group or a bridged cycloalkyl group; Qis —NO or —NO₂; and T is independently a covalent bond, a carbonyl, anoxygen, —S(O)_(o)— or —N(R_(a))R_(i)—, wherein o is an integer from 0 to2, R_(a) is a lone pair of electrons, a hydrogen or an alkyl group;R_(i) is a hydrogen, an alkyl, an aryl, an alkylcarboxylic acid, an arylcarboxylic acid, an alkylcarboxylic ester, an arylcarboxylic ester, analkylcarboxamido, an arylcarboxamido, an alkylaryl, an alkylsulfinyl, analkylsulfonyl, an arylsulfinyl, an arylsulfonyl, a sulfonamido, acarboxamido, a carboxylic ester, an amino alkyl, an amino aryl,—CH₂—C(T—Q)(R_(e))(R_(f)), or —(N₂O₂−)⁻·M⁺, wherein M⁺ is an organic orinorganic cation; with the proviso that when R_(i) is—CH₂—C(T—Q)(R_(e))(R_(f)) or —(N₂O₂−)·M⁺; then “—T—Q” can be a hydrogen,an alkyl group, an alkoxyalkyl group, an aminoalkyl group, a hydroxygroup or an aryl group.
 44. The composition of claim 37, wherein the atleast one compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor, or is a substrate for nitric oxide synthase is: (i) acompound that comprises at least one ON—O—, ON—N— or ON—C— group; (ii) acompound that comprises at least one O₂N—O—, O₂N—N—, O₂N—S— or —O₂N—C—group; (iii) a N-oxo-N-nitrosoamine having the formula: R¹R₂—N(O—M⁺)—NO,wherein R¹ and R² are each independently a polypeptide, an amino acid, asugar, an oligonucleotide, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbon, or a heterocyclic group, and M⁺ is an organic or inorganiccation.
 45. The composition of claim 44, wherein the compound comprisingat least one ON—O—, ON—N— or ON—C— group is an ON—O-polypeptide, anON—N-polypeptide, an ON—C-polypeptide, an ON—O-amino acid, an ON—N-aminoacid, an ON—C-amino acid, an ON—O-sugar, an ON—N-sugar, an ON—C-sugar,an ON—O-oligonucleotide, an ON—N-oligonucleotide, anON—C-oligonucleotide, a straight or branched, saturated or unsaturated,substituted or unsubstituted, aliphatic or aromatic ON—O-hydrocarbon, astraight or branched, saturated or unsaturated, substituted orunsubstituted, aliphatic or aromatic ON—N-hydrocarbon, a straight orbranched, saturated or unsaturated, substituted or unsubstituted,aliphatic or aromatic ON—C-hydrocarbon, an ON—O-heterocyclic compound,an ON—N-heterocyclic compound or a ON—C-heterocyclic compound.
 46. Thecomposition of claim 44, wherein compound comprising at least oneO₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— group is an O₂N—O-polypeptide, anO₂N—N-polypeptide, an O₂N—S-polypeptide, an O₂N—C-polypeptide, anO₂N—O-amino acid, O₂N—N-amino acid, O₂N—S-amino acid, an O₂N—C-aminoacid, an O₂N—O-sugar, an O₂N—N-sugar, O₂N—S-sugar, an O₂N—C-sugar, anO₂N—O-oligonucleotide, an O₂N—N-oligonucleotide, anO₂N—S-oligonucleotide, an O₂N—C-oligonucleotide, a straight or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted O₂N—O-hydrocarbon, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedO₂N—N-hydrocarbon, a straight or branched, saturated or unsaturated,aliphatic or aromatic, substituted or unsubstituted O₂N—S-hydrocarbon, astraight or branched, saturated or unsaturated, aliphatic or aromatic,substituted or unsubstituted O₂N—C-hydrocarbon, an O₂N—O-heterocycliccompound, an O₂N—N-heterocyclic compound, an O₂N—S-heterocyclic compoundor an O₂N—C-heterocyclic compound.
 47. The composition of claim 37,wherein the at least one compound that donates, transfers, or releasesnitric oxide, or induces the production of endogenous nitric oxide orendothelium-derived relaxing factor, or is a substrate for nitric oxidesynthase, is L-arginine, L-homoarginine, N-hydroxy-L-arginine,nitrosated L-arginine, nitrosylated L-arginine, nitrosatedN-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, citrulline,ornithine, glutamine, lysine, polypeptides comprising at least one ofthese amino acids or inhibitors of the enzyme arginase, cytokines,adenosin, bradykinin, calreticulin, bisacodyl, and phenolphthalein. 48.A method for producing platelets and/or proplatelets in vitro comprisingadding the composition of claim 37 to at least one megakaryocyte inculture.
 49. The method of claim 48, wherein the at least onemegakaryocyte in culture is obtained from a cell line, a stem cell, atissue, bone marrow, peripheral blood, a liver, a fetal liver or apatient's own blood megakaryocytic precursors.
 50. A method forproducing platelets and/or proplatelets in vivo in a patient in needthereof comprising administering to the patient an effective amount ofthe composition of claim
 37. 51. A method for treating or preventing ablood platelet disorder in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of thecomposition of claim
 37. 52. A method for treating or preventing a bloodplatelet disorder in a patient in need thereof comprising administeringto the patient a therapeutically effective amount of at least onethrombopoeisis stimulating factor and at least one compound thatdonates, transfers, or releases nitric oxide, or induces the productionof endogenous nitric oxide or endothelium-derived relaxing factor or isa substrate for nitric oxide synthase.
 53. The method of claim 52,wherein the compound is administered orally or by injection.
 54. Amethod for treating or preventing a blood platelet disorder in a patientin need thereof comprising: providing at least one megakaryocyte inculture; adding at least one thrombopoeisis stimulating factor and atleast one compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor or is a substrate for nitric oxide synthase to the atleast one megakaryocyte in culture to produce platelets and/orproplatelets; and administering a therapeutically effective amount ofthe platelets and/or proplatelets to the patient.
 55. The method ofclaim 54, wherein the thrombopoeisis stimulating factor is added to theat least one megakaryocyte prior to the step of adding the compound thatdonates, transfers, or releases nitric oxide, or induces the productionof endogenous nitric oxide or endothelium-derived relaxing factor or isa substrate for nitric oxide synthase.
 56. The method of claim 54,wherein the at least one megakaryocyte in culture is obtained from acell line, a stem cell, a tissue, bone marrow, peripheral blood, aliver, a fetal liver or a patient's own blood megakaryocytic precursors.57. The method of claimed 54, wherein the platelets and/or proplateletsare administered orally or by injection.
 58. The method of claim 17, 51,52 or 54, wherein the blood disorder is thrombc ytopenia, thrombocythmiaor thrombocytopathy.
 59. The method of claim 17 or 51, wherein thecomposition is administered orally or by injection.
 60. A compositioncomprising at least one megakaryocyte in culture and at least onecompound that donates, transfers, or releases nitric oxide, or inducesthe production of endogenous nitric oxide or endothelium-derivedrelaxing factor or is a substrate for nitric oxide synthase or apharmaceutically acceptable salt thereof.
 61. The composition of claim60, wherein the compound that donates, transfers, or releases nitricoxide, or induces the production of endogenous nitric oxide orendothelium-derived relaxing factor or is a substrate for nitric oxidesynthase is an S-nitrosothiol.
 62. The composition of claim 61, whereinthe S-nitrosothiol is S-nitroso-N-acetylcysteine, S-nitroso-captopril,S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine,S-nitroso-cysteine or S-nitroso-glutathione.
 63. The composition ofclaim 62, wherein the S-nitrosothiol is S-nitroso-glutathione.
 64. Thecomposition of claim 61, wherein the S-nitrosothiol is: (i)HS(C(R_(e))(R_(f)))_(m)SNO; (ii) ONS(C(R_(e))(R_(f)))_(m)R_(e); and(iii) H₂N—CH(CO₂H)—(CH₂)_(m)—C(O)NH—CH(CH₂SNO)—C(O)NH—CH₂—CO₂H; whereinm is an integer from 2 to 20; R_(e) and R_(f) are each independently ahydrogen, an alkyl, a cycloalkoxy, a halogen, a hydroxy, anhydroxyalkyl, an alkoxyalkyl, an arylheterocyclic ring, an alkylaryl, acycloalkylalkyl, a heterocyclicalkyl, an alkoxy, a haloalkoxy, an amino,an alkylamino, a dialkylamino, an arylamino, a diarylamino, analkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a sulfonic acid, asulfonic ester, an alkylsulfonic acid, an arylsulfonic acid, an.arylalkoxy, an alkylthio, an arylthio, a cycloalkylthio, a cycloalkenyl,a cyano, an aminoalkyl, an aminoaryl, an aryl, an arylalkyl, analkylaryl, a carboxamido, a alkylcarboxamido, an arylcarboxamido, anamidyl, a carboxyl, a carbamoyl, a carbamate, an alkylcarboxylic acid,an arylcarboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, acarboxylic ester, an alkylcarboxylic ester, an arylcarboxylic ester, ahaloalkoxy, a sulfonamido, an alkylsulfonamido, an arylsulfonamido, asulfonic ester, a urea, a phosphoryl, a nitro, —T—Q , or(C(R_(e))(R_(f)))_(k)—T—Q, or R_(e) and R_(f) taken together with thecarbons to which they are attached form a carbonyl, a methanthial, aheterocyclic ring, a cycloalkyl group or a bridged cycloalkyl group; Qis —NO or —NO₂; and T is independently a covalent bond, a carbonyl, anoxygen, —S(O)_(o)— or —N(R_(a))R_(i)—, wherein o is an integer from 0 to2, R_(a) is a lone pair of electrons, a hydrogen or an alkyl group;R_(i) is a hydrogen, an alkyl, an aryl, an alkylcarboxylic acid, an arylcarboxylic acid, an alkylcarboxylic ester, an arylcarboxylic ester, analkylcarboxamido, an arylcarboxamido, an alkylaryl, an alkylsulfinyl, analkylsulfonyl, an arylsulfinyl, an arylsulfonyl, a sulfonamido, acarboxamido, a carboxylic ester, an amino alkyl, an amino aryl,—CH₂—C(T—Q)(R_(e))(R_(f)), or —(N₂O₂—)⁻·M⁺, wherein M⁺ is an organic orinorganic cation; with the proviso that when R_(i) is—CH₂—C(T—Q)(R_(e))(R_(f)) or —(N₂O₂—)·M⁺; then “—T—Q” can be a hydrogen,an alkyl group, an alkoxyalkyl group, an aminoalkyl group, a hydroxygroup or an aryl. group.
 65. The composition of claim 60, wherein the atleast one compound that donates, transfers, or releases nitric oxide, orinduces the production of endogenous nitric oxide or endothelium-derivedrelaxing factor, or is a substrate for nitric oxide synthase is: (i) acompound that comprises at least one ON—O—, ON—N— or ON—C— group; (ii) acompound that comprises at least one O₂N—O—, O₂N—N—, O₂N—S— or —O₂N—C—group; (iii) a N-oxo-N-nitrosoamine having the formula: R¹R²—N(O—M⁺)—NO,wherein R¹ and R² are each independently a polypeptide, an amino acid, asugar, an oligonucleotide, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedhydrocarbon, or a heterocyclic group, and M⁺ is an organic or inorganiccation.
 66. The composition of claim 65, wherein the compound comprisingat least one ON—O—, ON—N— or ON—C— group is an ON—O-polypeptide, anON—N-polypeptide, an ON—C-polypeptide, an ON—O-amino acid, an ON—N-aminoacid, an ON—C-amino acid, an ON—O-sugar, an ON—N-sugar, an ON—C-sugar,an ON—O-oligonucleotide, an ON—N-oligonucleotide, anON—C-oligonucleotide, a straight or branched, saturated or unsaturated,substituted or unsubstituted, aliphatic or aromatic ON—O-hydrocarbon, astraight or branched, saturated or unsaturated, substituted orunsubstituted, aliphatic or aromatic ON—N-hydrocarbon, a straight orbranched, saturated or unsaturated, substituted or unsubstituted,aliphatic or aromatic ON—C-hydrocarbon, an ON—O-heterocyclic compound,an ON—N-heterocyclic compound or a ON—C-heterocyclic compound.
 67. Thecomposition of claim 65, wherein compound comprising at least oneO₂N—O—, O₂N—N—, O₂N—S— or O₂N—C— group is an O₂N—O-polypeptide, anO₂N—N-polypeptide, an O₂N—S-polypeptide, an O₂N—C-polypeptide, anO₂N—O-amino acid, O₂N—N-amino acid, O₂N—S-amino acid, an O₂N—C-aminoacid, an O₂N—O-sugar, an O₂N—N-sugar, 0₂N-S-sugar, an O₂N—C-sugar, anO₂N—O-oligonucleotide, an O₂N—N-oligonucleotide, anO₂N—S-oligonucleotide, an O₂N—C-oligonucleotide, a straight or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted O₂N—O-hydrocarbon, a straight or branched, saturated orunsaturated, aliphatic or aromatic, substituted or unsubstitutedO₂N—N-hydrocarbon, a straight or branched, saturated or unsaturated,aliphatic or aromatic, substituted or unsubstituted O₂N—S-hydrocarbon, astraight or branched, saturated or unsaturated, aliphatic or aromatic,substituted or unsubstituted O₂N—C-hydrocarbon, an O₂N—O-heterocycliccompound, an O₂N—N-heterocyclic compound, an O₂N—S-heterocyclic compoundor an O₂N—C-heterocyclic compound.
 68. The composition of claim 60,wherein the at least one compound that donates, transfers, or releasesnitric oxide, or induces the production of endogenous nitric oxide orendothelium-derived relaxing factor, or is a substrate for nitric oxidesynthase, is L-arginine, L-homoarginine, N-hydroxy-L-arginine,nitrosated L-arginine, nitrosylated L-arginine, nitrosatedN-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, citrulline,ornithine, glutamine, lysine, polypeptides comprising at least one ofthese amino acids or inhibitors of the enzyme arginase, cytokines,adenosin, bradykinin, calreticulin, bisacodyl, and phenolphthalein. 69.The composition of claim 60, wherein the at least one megakaryocyte inculture is obtained from a cell line, a stem cell, a tissue, bonemarrow, peripheral blood, a liver, a fetal liver or a patient's ownblood megakaryocytic precursors.
 70. A method for producing plateletsand/or proplatelets in vitro comprising adding the composition of claim60 to at least one megakaryocyte in culture.
 71. The method of claim 70,wherein the at least one megakaryocyte in culture is obtained from acell line, a stem cell, a tissue, bone marrow, peripheral blood, aliver, a fetal liver or apatient's own blood megakaryocytic precursors.72. A method for producing platelets and/or proplatelets in vivo in apatient in need thereof comprising administering to the patient aneffective amount of the composition of claim
 60. 73. A method fortreating or preventing a blood platelet disorder in a patient in needthereof comprising administering to the patient a therapeuticallyeffective amount of the composition of claim
 60. 74. The method of claim73, wherein the blood disorder is thrombocytopenia, thrombocythmia orthrombocytopathy.
 75. The miethod of claim 73, wherein the compositionis administered orally or by injection.
 76. The composition of claim 60,further comprising at least one thrombopoeisis stimulating factor. 77.The composition of claim 76, wherein the thrombopoeisis stimulatingfactor is interleukin 1, interleukin 2, interleukin 3, interleukin 4,interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin9, interleukin 10, interleukin 11, interleukin 12, interleukin 13,interleukin 14, interleukin 15, erythropoietin, thrombopoietin, stemcell factor, flt-3 ligand, granulocyte colony stimulating factor,granulocyte macrophage colony stimulating factor, tumor growth factorbeta, tumor necrosis factor alpha, interferon, fibroblast growth factor,platelet-derived growth factor, insulin-like growth factors, leukemiainhibitor factor, megakaryocyte colony stimulating factor, or mixturesthereof.
 78. The composition of claim 77, wherein the thrombopoeisisstimulating factor is thrombopoietin.
 79. A method for producingplatelets and/or proplatelets in vitro comprising adding the compositionof claim 76 to at least one megakaryocyte in culture.
 80. The method ofclaim 79, wherein the at least one megakaryocyte in culture is obtainedfrom a cell line, a stem cell, a tissue, bone marrow, peripheral blood,a liver, a fetal liver or a patient's own blood megakaryocyticprecursors.
 81. A method for producing platelets and/or proplatelets invivo in a patient in need thereof comprising administering to thepatient an effective amount of the composition of claim
 76. 82. A methodfor treating or preventing a blood platelet disorder in a patient inneed thereof comprising administering to the patient a therapeuticallyeffective amount of the composition of claim
 76. 83. The method of claim82, wherein the blood disorder is thrombocytopenia, thrombocythmia orthrombocytopathy.